Mechanisms of ligand induced downregulation and intracellular trafficking of the epidermal growth factor receptor.

Occupancy-induced downregulation of cell surface epidermal growth factor receptors (EGF-R) attenuates signal transduction. To define mechanisms through which downregulation of this class of growth factor receptors occurs, I have investigated the relative roles of ligand-induced internalization and recycling in this process. Occupied, kinase-active EGF receptors were internalized through a ligand induced, high affinity, endocytic system at rates up to 10-fold faster than empty receptors. In contrast, full length EGF receptors lacking tyrosine kinase activity underwent internalization at a rate independent of occupancy. This "kinase-independent" internalization rate appeared to reflect constitutive receptor internalization since it was similar to the internalization rate of both receptors lacking a cytoplasmic domain and of antibodies bound to empty receptors. EGF internalized by either kinase-active or kinase-inactive receptors was efficiently recycled and was found within endosomes containing recycling transferrin receptors. However, targeting of internalized receptors to lysosomes did not require receptor kinase activity. Targeting of internalized receptors to lysosomes was also independent of the domain required for ligand-induced internalization since receptors truncated at amino acid (a.a.) 958 were efficiently targeted to the lysosomes. The truncation at a.a. 958 eliminates a domain of the receptor required for ligand induced internalization. However, targeting to the lysosomes was dependent upon a domain between a.a.647-688 of the receptor. Receptors truncated at a.a.647 were unable to target to the lysosomes while those receptors extended to amino acid 688 were capable of lysosomal transfer. Only receptors that displayed ligand-induced internalization underwent downregulation, indicating that the proximal cause of downregulation is occupancy-induced endocytosis.

MECHANISMS OF LIGAND INDUCED DOWNREGULATION AND INTRACELLULAR TRAFFICKING OF THE EPIDERMAL GROWTH FACTOR RECEPTOR by John Herbst A dissertation subrnitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Pathology The IT niversity of Utah Deccrnbcr 1992 Copyright © John Joseph Herbst 1992 All Rights Reserved THE UNIVERSITY OF UTAH GRADUATE SCHOOL SUPERVISORY COMMITTEE APPROVAL of a dissertation submitted by J olm J. Herbst This dissertation has been read by each member of the following supervisory committee and by majority vote has been found to be satisfactory . 1/~4/~ 11~4AA ~h011~ { , J ' Lee Opresko THE UNIVERSITY OF UTAH GRADUATE SCHOOL FINAL READING APPROVAL To the Graduate Council of the University of Utah: I have read the dissertation of John J. Herbst in its final form and have found that (1) its format, citations, and bibliographic style are consistent and acceptable; (2) its illustrative materials including figures, tables, and charts are in place; and (3) the final manuscript is satisfactory to the supervisory committee and is ready for submission to The Graduate School. Date Chair: Supervisory Co Approved for the Department of Pathology John M. Matsen Chair Approved for the Graduate Council B. Gale Dick Dean of The Graduate School ABSTRACT Occupancy-induced downregulation of cell surface epidermal growth factor receptors (EGF-R) attenuates signal transduction. To define mechanisms through which downregulation of this class of growth factor receptors occurs, I have investigated the relative roles of ligand­induced internalization and recycling in this process. Occupied, kinase­active EGF receptors were internalized through a ligand induced, high affinity, endocytic system at rates up to lO-fold faster than empty receptors. In contrast, full length EGF receptors lacking tyrosine kinase activity underwent internalization at a rate independent of occupancy. This "kinase-independent" internalization rate appeared to reflect constitutive receptor internalization because it was similar to the internalization rate of both receptors lacking a cytoplasmic domain and of antibodies bound to empty receptors. EGF internalized by either kinase­active or kinase-inactive receptors was efficiently recycled and was found within endosomes containing recycling transferrin receptors. However, targeting of internalized receptors to lysosomes did not require receptor kinase activity. Targeting of internalized receptors to lysosomes was also independent of the domain required for ligand-induced intenlalization because receptors truncated at amino acid (a.a.) 958 were efficiently targeted to the lysosomes. The truncation at a.a. 958 eliminates a domain of the receptor required for ligand induced internalization. However, targeting to the lysosomes was dependent upon a domain between a.a.647- 688 of the receptor. Receptors truncated at a.a.647 were unable to target to the lysosomes while those receptors extended to amino acid 688 were capable of lysosomal transfer. Only receptors that displayed ligand­induced internalization underwent downregulation, indicating that the proximal cause of downregulation is occupancy-induced endocytosis. v CONTENTS ABSTRACT ...................................................................................... iv LIST OF FIGURES .......................................................................... viii Chapter 1. INTRODUCTION ..................................................................... 1 References ............................................................................... 22 2. THE ROLE OF LIGAND-INDUCED INTERNALIZATION IN DOWNREGULATION OF THE EPIDERMAL GROWTH FACTOR RECEPTOR ............................................................. 30 Summary ................................................................................ 31 Introduction ............................................................................ 32 Results .................................................................................... 34 Discussion ............................................................................... 45 Materials and Methods .............................................................. 50 References ............................................................................... 54 3. THE ROLE OF TYROSINE KINASE ACTIVITY IN RECYCLING AND COMPARTMENTALIZATION OF THE EPIDERMAL GROWTH FACTOR RECEPTOR ....................... 57 Summary ............................................................................... 58 Introduction ........................................................................... 59 Results ................................................................................... 62 Discussion .............................................................................. 76 Materials and Methods ............................................................. 78 References .............................................................................. 82 4. SEQlTENCES WITHIN THE EPIDERMAL GROWTH FACTOR RECEPTOR THAT DIRECT LYSOSOMAL TARGETING ......... 87 Summary ................................................................................ 88 Introduction ............................................................................ 90 Results .................................................................................... 92 Discussion ............................................................................. 111 Materials and Methods ............................................................ 118 References ............................................................................. 123 5 . SUMMARy ........................................................................... 127 References ............................................................................. 138 Vll LIST OF FIGURES Figure 2.1. Internalization and Downregulation of EGF Receptors .................. 36 2.2. Transferrin Internalization in B 82 cells Expressing Human EGF Receptors ............................................................... 39 2.3. TPA Inhibition of Ligand-Induced EGF Receptor Internalization ...................................................... 41 2.4. Effect of TPA on the Rates of EGF andTransferrin Receptor Internalization ....................................... 43 2.5. Effect of TP A on A654 EGF Receptor Internalization ................... 44 2.6. Effect of TPA on EGF-induced EGF Receptor Downregulation ...... 46 3.1. Inside/Surface Ratios of EGF in WT and Kinase Negative B82 cells ........................................................... 64 3.2. Internalization of WT and Kinase Negative EGF Receptors at Steady State ..................................................... 65 3.3. Recycling of EGF and Transferrin in B82 Cells ............................. 67 3.4. Colocalization of EGF and Transferrin in Recycling Endosomes ..... 69 3.5. Colocalization of EGF and Transferrin in Recycling Endosomes ..... 72 3.6. HRP-transferrin Conjugates can Density Shift Internalized EGF in B 82 Cells .................................................... 74 4.1. Downregulation of EGF Receptors ............................................... 94 4.2 Accumulation and Degradation of Internalized EGF ....................... 96 Figure Page 4.3. Internalization of WT and Kinase Negative EGF Receptors at Steady State .................................................... 99 4.4. Relative Amounts of Recycling and Degradation of EGF .............. 102 4.5 Internalization of WT, Kinase Negative, i\647 , i\958 Kinase Positive and i\958 Kinase Negative EGF Receptors .... 104 4.6. Accumulation of EGF within Lysosomes Requires the Cytoplasmic Domain of the EGF Receptor ............................. 106 4.7. The Domain Between a.a. 647-688 of the EGF-R is Required for Degradation ...................................................... 1 08 4.8. In/Sur Ratios of Occupied and Unoccupied EGF-R ....................... 110 4.9. The Effect of Chase Conditions on Degradation .......................... .112 4.10 Transfer of EGF to Lysosomes is a Saturable Process .................. 113 5.1 A Model of EGF Receptor Internalization and Sorting to SpecificIntracellular Compartments .............................. 135 ix ACKNOWLEDGMENTS I wish to express my deepest gratitude to my major professor Dr. Steven Wiley. His excellent example as a scientist and friend furthered my education and development as a scientist. He created a superior learning environment providing the perfect mix of freedom and direction. Most of all Steve was always there when I needed him and he was always willing to help. I also wish to thank Dr.Lee Opresko, Mrs. Brenda Walsh and Dr. Kirk Lund for their patience and friendship. They provided a standard of laboratory goodwill and harmony that will be hard to equal. I want to thank the members of my committee, Dr. Steven Wiley, Dr.Lee Opresko, Dr. David Low, Dr. Raymond Daynes, and Dr. Martin Rechsteiner, for their support and guidance throughout my graduate career. Most of all I wish to thank my parents without whose support and guidance my education would have been impossible. Part of this research was funded by Public Health Service, National Research Service A ward Training Grant 5 T32 CA 09097. CHAPTER 1 INTRODUCTION 2 The membrane barrier of cells is essential for life itself. It maintains the differences between the interior of the cell and the external environment. The lipid composition of the plasma membrane was recognized in 1899 by E. Overton. Overton's experiments on the penetration of hundreds of chemical compounds into plant and animal cells demonstrated that only those compounds that were lipid soluble were able to penetrate into the cells. Overton's hypothesis was confirmed by chemical isolations and characterizations demonstrating that a typical content of major lipid in an erythrocyte plasma membrane, expressed as percent of total lipid by weight, is 23% cholesterol, 18% phosphatidylethanolamine, 7% phosphatidylserine, 17% phosphatidylcholine, and 18% sphingomyelin.(I) The organization of the lipid to form a membrane remained a mystery until 1925 when Gorter and Grendel performed their classic experiments to determine the bilayer structure of the lipid membrane.(2) These experiments consisted of extracting the lipid from erythrocytes of dogs, sheep, rabbits, guinea pigs, goats, and man and then determining the surface area these extracted lipid occupied and comparing this area to the area of intact erythrocytes. Using this method they determined that the extracted lipid occupied an area approximately twice that of the intact erythrocytes, leading them to propose the bilayer nature of the lipid membrane. Further advances into the structure of the plasma membrane include the assaying of the fluid mobility of the membrane by Frye and Eddin, and Poo and Cone, and the identification of membrane spanning 3 proteins by Bretscher.(3-5) All of these experiments, and many others, led to the formation of the fluid mosaic model of Singer and Nicholson that is the basis for all models of membrane structure today.(6) This model states that the plasma membrane consists of a lipid bilayer in which proteins may be either associated with the external or internal faces of the plasma membrane, or completely span the plasma membrane as integral membrane proteins. However, whether these proteins span the complete lipid bilayer or not, all are theoretically capable of freely diffusing within the plane of the membrane. Several functional requirements for the plasma membrane are obvious. First, the cell must have a nleans to regulate intracellular ion concentrations. Second, the cells must have a means to ingest essential nutrients as well as a means of eliminating toxic byproducts from the metabolism of these nutrients. Finally, cells must have a means of perceiving their environment and communicating with the other cells that surround them. Cells have developed a number of different mechanisms to deal with these problems. Hydrophobic molecules and small uncharged polar molecules such as water are freely diffusible across the plasma membrane. Other biologically impoltant molecules can diffuse across the plasma membrane using carrier proteins or ion channels, so long as they are diffusing down a concentration gradient, in a process called facilitated diffusion. Alternatively cells can use a process called active transport to move these ions against a concentration gradient at the expense of energy 4 in the form of adenosine triphsophate A TP.(7 -9) Both of these processes are used extensively by cells and provide effective mechanisms for the transport of molecules across the cell membrane. The limitation of these processes is that they are generally restricted to the transport of low molecular weight proteins. To overcome the size obstacle, cells have evolved another process, called endocytosis, to transport material across the cell membrane. Endocytosis is divided into two processes both of which ingest material from the extracellular space and deliver it to various compartments within the cell.(10) The first process, called phagocytosis (from the Greek word phagien meaning to eat), is a mechanism to ingest large particulate matter. The second mechanism is designed for the intake of fluid material and is called pinocytosis ( from the Greek word pineain meaning to drink). Phagocytosis has been studied extensively in the protozoa where this process has an obvious nutritive role. In multicellular animals phagocytosis was first observed in macrophages where these cells ingest foreign particulate matter and deliver them to the lysosome for degradation. Macrophages were also the first cell type in which pinocytosis was observed. W.H. Lewin was the first to describe this process in 1931 when using time lapse photography and light microscopy he observed the formation of vesicles approximately 1-2Jlm in diameter from areas of the cell that displayed vigorous cytoplasmic motion. These vesicles appeared to engulf and subsequently transport fluid into the 5 interior of the celL(11-13) Advances in microscopy, particularly the development of the electron microscope, led to the discovery of pinocytotic vesicles that were much smaller (ranging down to O.lJlm in diameter) as well as the realization that this endocytic process occurred not only in phagocytic cells, such as macrophages, but in all animal celIs.(14,15) Studies using either fluorescent nlarkers or electron dense material such as horseradish peroxidase later confirmed that these vesicles were indeed the site of fluid uptake.(14,16) These studies also demonstrated that pinocytosis is a constitutive process and that the amount of material taken up is proportional to the concentration of that material in the extracellular environment. This constitutive pinocytic uptake can be appreciable. Mouse L cells, for example, take up an amount of membrane equal to 50% of its entire surface every hour. If synthesis were the only replacement for this membrane, then the cells would quickly consume themselves. Thus there must be a mechanism for recycling of internalized membrane. Pinocytosis solves the problem of transporting large molecules that are impervious to the plasma membrane into the cell. Pinocytosis also has the advantage over active and facilitated transport in that it can deliver these molecules to discreet compartments within the cell. However even with both of these advantages, pinocytosis has two major limitations. First, it is nonselective. Pinocytosis internalizes everything that is soluble in the extracellular milieu. Second, pinocytosis is inefficient. Pinocytosis is only capable of internalizing molecules in proportion to their concentration in the extracellular milieu. To 6 overcome these limitations cells have evolved receptors that specifically bind a molecule with high affinity and then internalize it in a process called receptor mediated endocytosis. Receptor mediated endocytosis's main advantages over pinocytosis are the specificity and the affinity with which the receptors bind ligand. These dual qualities of high specificity and high affinity allow the cells to perceive and utilize these molecules even when they are present at extremely low concentrations. To date, more than 50 different ligands have been found to bind to specific receptors at the cell surface. Receptors have been discovered for such diverse molecules as low density lipoprotein (LDL), transferrin, and vitellogenin; as well as insulin, acetylcholine, and epidermal growth factor EGF). After careful examination of the physiologic properties of the known receptors and their ligands Kaplan proposed categorizing the receptors based upon their physiology.(17) According to this scheme for classification, receptors can be grouped into one of two classes. Class I receptors are those receptors whose main function is to transmit information to cells. These receptors include the growth factor and hormone receptors such as the insulin and the epidermal growth factor receptor (EGF-R). Class II receptors are those receptors whose primary function is to mediate the transport of metabolically significant molecules into the cell for further processing. Examples of this class of receptors include the transferrin receptor and the LD L receptor. Receptors within the same class tend to share similar requirements for ions in binding of their ligands. They also have similar patterns of surface distribution, receptor reutilization and regulation. 7 Kaplan's classification, based on physiology, also serves to demonstrate that although receptors are generally specific for a single ligand and possess high affinity for these ligands (disassociation constants as low as 1 x 10-12), it has been the physiologic properties that have led to their discovery. For example, the LDL receptor was discovered because of the inability of patients with familial hypercholesterolenlia to clear LDL from their blood.(18,19) Over 50 ligands have been found to enter the cell by receptor mediated endocytosis. All of these systems share many common features. Brown, Anderson and Goldstein have proposed that four common features in all of these systems define receptor mediated endocytosis.(20) The first of the four properties carefully defines what is meant by a receptor. According to this definition a receptor must bind an exogenous ligand and in doing so achieve a physiological effect. This definition distinguishes receptors for receptor mediated endocytosis from receptors for foreign molecules such as toxins and viruses. The second property that defines receptor mediated endocytosis is that once the ligand has bound to the receptor, the internalization of the receptor ligand complex must be rapid. The half life for internalization of the receptor ligand complex on the cell surface must be under 10 min. The third requirement is that the receptor ligand complex enter the cell through a coated pit. Finally, the fourth requirement is that the internalized ligand 8 be delivered to a specific intracellular compartment. Usually this compartment is the lysosome. There are however several notable exceptions to the rule regarding lysosomal delivery. Yolk proteins, such as vitellogenin, concentrate in yolk granules without degradation.(20,2l) Maternal immunoglobulins are transcytosed across the intestinal epithelium thus conferring passive immunity to the fetus.(22-26) Finally, nerve growth factor is internalized at the tip of the axon and migrates to the nerve cell body where it accumulates.(27) These characteristics also define the four steps involved in receptor mediated endocytosis. These steps are binding of the ligand to its receptor, clustering of the receptor in coated pits, internalization of the receptor, and intercellular targeting of the internalized ligand. Many receptors require divalent cations such as calcium to bind their ligands. These receptors may also be sensitive to low pH which causes disassociation of the ligand from the receptor. The receptors that are dependent upon divalent cations and are pH sensitive are almost exclusively class II receptors. These receptors also undergo reutilization in the form of recycling of the receptor to the plasma membrane. Thus the requirement for divalent cations and the sensitivity of the ligand binding to low pH could playa critical role in the disassociation of the ligand from the receptor and the subsequent delivery of the ligand to the lysosome while the receptor is recycled back to the cell surface. Examples of these receptors are the LDL receptor, the vitellogenin receptor, and the alpha 2 macroglobulin receptor.(27-33) 9 Once the receptor has bound ligand it must then enter a coated vesicle in order to be efficiently internalized. The name coated vesicles refers to the fuzzy appearance of the vesicles when viewed by microscopy. The role of coated vesicles in endocytosis was first recognized by Roth and Porter when they correlated the uptake of mosquito yolk proteins to these coated vesicles.(34) Pearse was the first to isolate the major protein component of these coats identified as a 180,000 MW nonglycosylated protein named clathrin.(35,36) In addition to the 180,000 MW clathrin molecule several "clathrin associated proteins" have been isolated. The most predominant of these are a pair of 35,000 MW polypeptides called the clathrin light chains. Coated vesicles derived from brain also show bands at 100-110,000 MW and 55,000 MW as analyzed by sodium dodecylsulfate (SDS) gels.(37,38) Clathrints location on the cytoplasnlic face of coated vesicles is based on several lines of evidence. The first comes from electron micrographs which visually localize the clathrin to the cytoplasmic face of budding vesicles.(38,39) Biochemical evidence for the cytoplasmic localization was obtained using isolated coated vesicles. In these isolated vesicles the clathrin is sensitive to added proteases and is easily dissociated by mild treatment such as 0.02M Tris chloride pH 7.5, or 2M Urea, neither of which would be expected to disrupt the lipid bilayer.(42,43) Coated pits comprise about 20/0 of the cell surface.(23) Goldstein and Brown calculated the lifetime for the coated pit at 1 min and the turnover of the entire cell surface every 15 min.(41) This short half-life, the rapid turnover, and the fact that coated pits do not seem to be depleted 10 strongly argue for recycling of plasma membrane as well as recycling of the clathrin molecules. Further evidence for the direct recycling of the clathrin from internalized vesicles is provided in the fact that no intracellular pools of clathrin have been found. (42-44 ) The importance of clathrin coated pits in receptor mediated endocytosis is now a generally accepted hypothesis. Coated pits have been shown to mediate the internalization of both class I and class II receptors such as LDL, EGF, alpha 2 macroglobulin, and insulin.(20,4S-47) Additional support for this hypothesis is the observation that LDL receptors that are deficient in their ability to be internalized fail to cluster in coated pits. Furthermore, when cells are depleted of intracellular potassium coated pit formation and receptor mediated endocytosis are arrested.(20,48) Coated pits also appear to concentrate some plasma membrane proteins and exclude others. Certain ligands such as LD L are known to be preclustered in coated pits. Others, such as EGF-Rs, are randomly distributed on the cell surface and only cluster in the coated pits after ligand binding.( 49) Other membrane proteins appear to be excluded from the coated pits. An example of this is the surface antigen Thy-l which are present in coated pits at 1 % of the level that would be expected from random distribution.(SO) These observations have led scientists to postulate that the clathrin acts as a molecular sieve concentrating some proteins to facilitate their rapid internalization while excluding other to increase their half life on the surface of the cell. However, some controversy as to the exact role clathrin coated pits play 11 in internalization and intracellular trafficking exists as yeast mutants that lack the gene for clathrin are still able to secrete proteins normally .(51) One postulate of the hypothesis that efficient internalization requires association of the receptor with coated pits is that in certain situations, the number of receptors should exceed the number of binding sites for the receptors in coated pits. If this situation occurred, then the number of binding sites in the coated pits would become the rate limiting factor and the internalization in these cells would be less efficient. Anderson, Brown and Goldstein found exactly this situation in A431 cells.(54) A431 cells contain 10-fold more LDL receptors than other fibroblastic cell lines. These receptors have the same affinity and specificity for LDL as receptors in other cell lines, but were 10-fold less efficient in their ability to internalize LDL. A similar situation was discovered by Wiley when examining the effect of ligand concentration on the specific internalization rate (ke) of the EGF-R in A431 cells. He found that as the concentration of receptor-ligand complexes on the cell surface increased, their ke decreased. Additionally he determined that the increasing concentrations of EGF ligand receptor complexes had no effect on the internalization rate of the transferrin receptor.(53) These results together with those of Anderson et aL indicate that even though coated pits are essential for the internalization of receptors, and although the EGF-R, the transferrin receptor, and the LDL receptor are all internalized via coated pits, they must bind to different determinants. 12 Two basic approaches have been used to study the internalization of receptors. Both approaches involve labeling the ligand for the receptor and then following the receptor-ligand complex. The first approach involves following the trafficking of the ligand visually. Using this approach the ligands are tagged with a marker that can be visualized directly, such as ferritin or a fluorescent compound, or enzymatic tags that can be stained histochemically and then visualized. The advantage of this approach is that structures through which the ligand receptor complex traverses can be visualized. Additionally, by using dual tags other membrane receptors that may be internalized together may be detected. This approach has proved extremely useful. It has been used to study the LDL system and to identify coated pits as the structure involved in the internalization of this molecule.(20) These visual probes have also been used extensively to identify the components that are internalized within the coated pit structure, such as colocalizing of EGF and LDL receptors.(54) Epidermal growth factor receptor ligand-induced clustering within coated pits and subsequent internalization has also been visualized using these techniques.(55-57) Additionally, this approach has shown the structures involved in intracellular trafficking of the receptor following internalization. Internalized vesicles were found to cluster in the perinuclear region prior to lysosomal degradation and has implicated the process of endocytosis and subsequent exocytosis at the leading edge of cells in cellular locomotion. (46,49,58) The limitation of this approach in studying internalization is that the data are somewhat 13 subjective and qualitative. The kinetic approach is another way to study of internalization. These studies typically involve the following of a radio labeled ligands interactions with cells at either steady state conditions or as the cells approach steady state. This approach has the advantage of being quantitative and thus yields rate constants for internalization, recycling, degradation, insertion of new receptors and trafficking of the receptors between distinct compartrnents.(59-62) Using these constants it becomes possible to distinguish between different mathematical models of receptor behavior as well as examine the effects of modulators, such as tumor promoters or binding of other ligands, on endocytic behavior. For example, kinetic analysis can distinguish between a receptor that is not internalized and one that is rapidly recycled. Using visual assays the results would appear identical, as the majority of receptors would remain at the cell surface. Combining both of these approaches with the power of molecular biology has revealed specific sequences in the cytoplasn1ic tails of receptors that are necessary for internalization. A specific mutation at residue 807 which converted tyrosine to cysteine in the LDL receptor renders that receptor internalization defective.(63) Lazarovirs and Roth have reported that conversion of a specific cysteine to tyrosine at position 543 in the normally non internalizing influenza virus hemagglutinin renders this protein capable of being internalized by coated pits.( 64) The importance of tyrosine residues in internalization can also been extended to the transferrin receptor, the polymeric immunoglobulin 14 receptor and the mannose-6-phosphate receptor.(65-68) The requirement for a tyrosine may not be a universal signal for internalization as no consensus sequence has yet been found for internalization. Indeed, there is no obvious sequence homology between the transferrin and LDL receptor cytoplasmic domains required for internalization.(63) However, transferrin and EG F receptors do not compete for entry into the coated pit pathway for internalization.(69) These results support the idea that there may be separate binding sites for internalization and therefore separate internalization sequences in coated pits. The intrinsic kinase activity of some class I receptors has been reported to be essential for their ligand-induced internalization. The insulin receptor's kinase activity is essential for ligand-induced internalization.(70-73) The internalization of the EGF-R is also dependent upon receptor tyrosine kinase activity.(73) However, the absolute necessity of kinase activity remains uncertain as truncations of the EGF-R can eliminate the dependency upon kinase activity.(74) Kinase activity may not directly mediate internalization of class I receptors. Instead the phosphorylation of accessory proteins may stabilize binding of specific regions of the cytoplasmic tail allowing for efficient internalization. However, because other class I receptors do not possess kinase activity and because there is no sequence homology for an internalization domain the structural determinants required for internalization remain unclear. Transferrin receptors, LDL receptors, and EGF receptors have all 15 been shown to internalize via coated pits. Once internalized these receptors share quite different fates. The transferrin receptor and the LDL receptor are recycled extensively whereas the EGF-R is extensively degraded. The identity of the compartments, the number of compartments, and how the trafficking between these compartments is controlled, have been the subject of extensive research. One method used to investigate the intracellular trafficking of receptors has been to follow a radiolabeled ligand as it progresses through the cell. As the ligand traverses the cellular compartments it can be separated by differential centrifugation through density gradients. These compartments are then analyzed using enzymatic markers to identify the compartment as being lysosomal, golgi, or mitochondrial.(75-78) One elegant refinement of this process is the use of horseradish peroxidase and diaminobenzidine to shift the density of endocytic compartments.(79) When diaminobenzidine is exposed to peroxidase it will cross-link and form an insoluble complex of higher density. Aijoka and Kaplan improved this protocol by conjugating horseradish peroxidase to transferrin, thereby allowing them to follow a specific ligand through the endosomal compartments. They used this technique to demonstrate that EGF and transferrin are internalized into the same endosome and that internal pools of transferrin receptors result from the constitutive internalization of transferrin receptors.(80,81) Another approach to studying intracellular trafficking is to dissect the degradative processing of a ligand by the use of lysosomotropic 16 agents.(82,83) An example of this type of analysis can be seen in Wiley's work on the intracellular processing of EGF. Wiley identified four steps in the processing of EGF. These steps showed differential sensitivity to lysosomotropic agents such as monensin, leupeptin, methylamine and low temperature. (84) Through the use of these techniques, as well as others such as electron microscopy, it has become generally accepted that an early endosomal compartment called CURL (compartment for uncoupling of receptor and ligand) is the site at which sOlting of receptors for either recycling or degradation occurs.(85) One model of sorting states that vesicles that form from tubular extensions of the CURL are sorted to the plasma membrane whereas the remainder of the sorting endosome is delivered to the lysosome.(86) This model predicts that an unoccupied receptor, or a receptor containing no sorting information, would partition to the tubular extensions of the CURL which contain 10-fold more membrane and thus have more surface area.(87) Conversely, occupied receptors that contain a sorting signal are retained within the cisternal elements of the sorting endosome by specific anchoring to some secondary sorting protein or structure. One implication of this model is that binding of a ligand to its receptor exposes a site on the receptor that allows it to bind to the sorting protein. Studies using the LDL receptor have revealed that if monensin is used to prevent the acidification of the endosome, and thus prevent ligand dissociation, recycling of the receptor is strongly inhibited.(88) This implies that unoccupied and occupied receptors are sorted differently. 17 Similar studies involving the use of agents to raise the intracellular pH, and thus prevent ligand dissociation, have shown that the recycling of these insulin receptors is inhibited.(89) Studies on the kinetics of insulin receptor dissociation and degradation have shown a temporal relationship between ligand dissociation from the receptor and subsequent degradation. The amount of insulin receptor loss was also correlated to the amount of insulin receptor that remained bound to its ligand.(90) A similar correlation has been made to account for the differences in endocytic processing between proinsulin and insulin.(91) EGF and TGF-alpha both bind the EGF-R, yet TGF-alpha has quantitatively higher levels of biological activity than EGF. The differences in biological activity can be correlated to TGF- alpha's inability to be degraded and its subsequent recycling in an intact biologically active form by cells.(92) TGF-alpha was also incapable of completely downregulating the EGF-R. The inability of TGF- alpha to be degraded, and the inability of the EGF-R to be downregulated by TGF­alpha was proposed to be due to its dissociation from the EGF-R at a much higher pH than EGF. After internalization the TGF-alpha rapidly dissociates from the EGF-R, returning the EGF-R to a conformation that conceals the sorting sequence and allowing the recycling of both ligand and receptor.(92) Occupancy of receptors has also been shown to direct the intracellular trafficking of vesicles by blocking vesicular fusion.(93) Carbohydrate structure of ligands has also been implicated in intracellular 18 targeting in the GaVGal NAC receptor system. The GaVGal NAC receptor binds both asialoorosomucoid (ASOR) and asialotriantennary glycopeptide(TRI). TRI dissociates fronl the receptor while ASOR does not. In perfused rat liver 52% of the internalized TRI was released intact while 98% of the ASOR was degraded.(94) Further evidence for protein sorting signals comes from the study of polarized epithelium in hepatocytes. Hepatocytes are a polarized cell with apical and basolateral plasma membrane domains with distinct protein compositions. During membrane biogenesis all membrane proteins are shipped first to the basolateral domain. Apical proteins are then internalized and transcytosed to the apical surface.(95) This vectorial transport implies targeting sequences within the apical proteins to direct their transport and specialized proteins to recognize these sequences. Indeed, differences in the composition of apical and basolateral transport vesicles have been reported.(96) One of the most widely used models for protein sorting is the MOCK cell. Golgi to basolateral sorting in these cells has been found to involve cytoplasmic determinants in several proteins. The polymeric imnlunoglobulin receptor ( plgR) requires a determinant in the cytoplasmic tail that allows entry into coated pits.(97) This domain was further elucidated by Casanova et al. to the phosphorylation of a serine residue at position 664. However, it is the negative charge and not the phosphate itself that is responsible for transcytosis as replacement of the serine with aspaitic acid to mimic the charge of the phosphate group 19 resulted in a receptor that behaved identically to the wild type receptor.(98) A model consistent with these data is that the negative charge of the phosphate group alters the receptor conformation exposing a sorting signal. Not all sorting signals are similar to those required for clustering in coated pits and efficient internalization. In the LDL receptor the sorting sequence is independent of the critical tyrosine required for clustering and internalization. (99,100) Thus, although several lines of evidence support the existence of protein sorting sequences within the cytoplasmic tail of membrane proteins, the issue remains unclear as no consensus sequence or motif has been discovered. Receptor mediated endocytosis and intracellular trafficking are complex processes that are under extensive regulatory control. Sequences contained in the receptors themselves are required for normal interaction of the receptor with the cellular machinery for internalization and sorting. The exact nature of these sequences remains unclear. Loss of the ability of the receptors to interact with the machinery for endocytosis and intracellular trafficking can have dire consequences for the organism as is seen with mutations in the LDL receptor leading to hypercholesterolemia. In these studies I will explore the sequences required for internalization and intracellular trafficking of the polypeptide hormone receptor for EGF and how alterations in either internalization or intracellular trafficking affect regulation of the 20 receptor at the level of downregulation. Polypeptide hormones are essential for the growth and survival of all animal cells. EGF is a polypeptide hormone that acts as a mitogen for a variety of cell types, both in vitro and in vivo.(101-103) Overexpression of the proto-oncogene erbB EGF receptor (EGF-R) has been seen in vulval tumors, as well as tumors from breast, ovary cervix and kidney.(104) One study has shown that a reduction in the number of EGF-R's expressed on A431 cells resulted in a diminution of tumorigenicity when these cells were injected into in athymic mice.(105) Another study has shown transformation of cells by receptors that are unable to be internalized and thus can not be degraded.(106) The EGF-R, like other hormone receptors, is under extensive regulatory control. This extensive regulation presumably contributes to normal growth control of the cell. Abrogation of regulatory controls could contribute to uncontrolled growth as seen in the constitutively active erbB transformed cells, and EGF-R overexpression in human tumors. One of the most ubiquitous regulatory control mechanisms observed in hormone receptor literature is "downregulation" of the receptor subsequent to ligand addition.(107-109) Although downregulation has been observed in a number of other receptor systems, such as the LDL receptor system, EGF appears to differ from these systems in that the downregulation does not occur through a modulation of the synthetic rate of the receptor. ( 104) Thus even though downregulation of the EGF-R is recognized to be an extremely important event in the regulation of cellular responses to EGF, the mechanistic basis 21 for this process remains poorly understood and controversial.( 111,112) In the context of this study, downregulation is defined as a shift in the pool of EGF-R's from the surface of the inside of the cell and a subsequent increase in receptor degradation rates. This redistribution serves to negatively attenuate the the signal transduction of EGF by limiting the number of receptors at the cell surface that can be occupied to generate the cellular responses that occur in the presence of EGF.(110) EGF-R downregulation could be occurring through selective internalization of occupied versus unoccupied receptors. It could also occur through selective recycling of unoccupied versus occupied receptors, selective degradation or enhanced degradation of the EGF-R in the presence of ligand, or a combination of all of these processes. All of these processes can theoretically regulate the receptor distribution within the cell and can be broken down into two basic mechanisms: those that regulate receptor entry into the endocytic pathway and those that target the internalized receptors for recycling or degradation in the lysosomes. Alterations in either of these two pathways could have profound effects on the ability of the cell to attenuate the response of the cell to EGF as well as alter the ability of the EGF-R to interact with key intracellular substrates for second messenger generation. The purpose of my investigation is to determine which aspects of endocytosis and intracellular trafficking (ligand-induced internalization, recycling, and degradation) are essential for EGF induced EGF-R downregulation and what information within the EGF-R itself is necessary to direct the regulation of receptor distribution. In order to 22 accomplish this I have used mouse B-82 cells that lack endogenous EGF­Rls but have been transfected with both the full length EGF-R and various point mutations and truncations of the EGF-R. These cells were then screened for their ability to downregulate to find a mutant that lacked this ability. Once a mutant was located that lacked the ability to downregulate it was compared to the wild type receptor in the parameters of internalization, recycling, degradation and intracellular trafficking to determine where these receptors differed. My experiments sought to determine: 1. What information within the EGF-R itself is necessary for the cell to be able to effectively downregulate the receptor. 2. How specific mutations affect the receptors ability to interact with the cellular machinery of endocytosis and intracellular trafficking. This goal will include asking the following questions; a. Does a specific mutation abrogate the receptors ability to be efficientl y internalized? b. Does a specific mutation block ligand accelerated degradation indicating that this is necessary for downregulation? c. Does a specific mutation affect receptor recycling? Are the mutated and the wild type receptor recycled through the same pathway or are there multiple pathways for recycling? References 1) Sheeler, P. and Bianchi, D.E. (1983) Cell Biology: Structure, Biochemistry, and Function. Wiley and Sons pubs. pp.327-339. 2) Gorter, E., and Grendel, F. (1925) J. Exp. Med.41, 439-443. 23 3) Frye, L.D., and Edidin, M. (1970) J. Cell Sci. 7, 319-335. 4) Poo, M., and Cone, R.A. (1974) Nature 247, 438-441. 5) Bretscher, M.S. (1971) J. Mol. BioI. 59, 351-357. 6) Singer, SJ., and Nicolson, G.L. (1972) Science 175, 720-731. 7) Tanford, C. (1978) Ann Rev. Biochem. 47, 933-965. 8) Kyle, J. (1981) Nature 292, 201-204. 9) Sweadner, K.J., and Goldin, S.M. (1980) N EJ.Med.302, 777. 10) Silverman, S.C., Stienman, R.M., and Cohn, Z.A. (1977) Ann. Rev. Biochem. 46,669-722 11) Holter, H. (1959) Int. Rev. Cytol. 8, 481-504. 12) Anderson, R.G., and Kaplan, J. (1983) Modem Cell BioI. 1, 1-52. 13) Lewis, W.H. (1931) Bull. Johns Hopkins Hosp. 49,117-136. 14) Silverstein, S.C., Steinman, R.M., and Cohn, Z.A. (1977) Ann. Rev. Biochem. 46, 669-772. 15) Palade, G.E. (1956) J. Biophys. Biochem. Cytol. Z (Suppl) 85-88. 16) Steinman, R.M., and Cohn, Z.A. (1972) J. Cell BioI. 55 ,186-204. 17) Kaplan, J. (1981) Science 212, 14-20. 18) Goldstein, J.L., and Brown, M.S. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 2804-2808. 19) Brown, M.S., and Goldstein, J.L. (1986) Science 232, 34-47. 20) Goldstein, J.L., Anderson, R.G., and Brown, M.S. (1979) Nature 279, 679-685. 21) Opresko, L.K., and Karpf, R.A. (1987) Cell 51, 557-568. 24 22) Rodewald, R. (1973) J. Cell BioI. 58,187-211. 23) Linen, C.D., and Roth, T.F. (1978) J. Cell Sci. 33,317-328. 24) Abrahamson, D.R., Powers, A., and Rodewald, R. (1979) Science 206, 567-569. 25) Abrahamson, D.R., and Rodewald, R. (1981) J. Cell BioI. 91, 270- 280. 26) Balcarova-Stander, J.S., Pfeiffer, S.E., Fuller, S.D., and Simons, K. (1984) E.M.B.O. 3, 2687-2694. 27) Bradshaw, R.A. (1978) Ann. Rev. Biochem. 47, 191-216. 28) Brown, M.S., and Goldstein, J.L. (1975) Cell 6, 307-316. 29) Brown, M.S., Goldstein, J.L., and Anderson, R.G.W. (1977) Cell 10, 351-364. 30) Opresko, L.K., and Wiley H.S. (1987) J.BioI. Chern. 262,4109- 4115. 31) Kaplan, J. (1980) Cell 19, 197-205. 32) Kaplan, J., and Nielson, M.N. (1979) J. BioI. Chern. 254, 7322- 7328. 33) Wallace, R.A., and Jared, D.W. (1975) J. Cell BioI. 69, 345-351. 34) Roth, T.F., and Porter, K.R. (1964) J. Cell BioI. 70, 313-332. 35) Pearse, B.M.F. 91976) Proc. NatI. Acad. Sci. U.S.A. 73, 1255- 1259. 36) Pearse, B.M.F. (1975) J. Mol. BioI. 97, 93-98. 37) Harrison, S.C., and Kirchhausen, T. (1983) Cell 33, 650-652. 38) Pearse, B.M., and Bretscher, M.S. (1981) Ann. Rev. Biochem. 50, 85-101. 25 39) Blitz, A.L., Fine, R.W., and Toselli, P.A. (1977) J. Cell BioI. 75, 135-147. 40) Schook, W., Puszkin, S., Bloom, W., Ores, C., and Kochwa, S. (1979) Proc. Nati. Acad. Sci. U.S.A. 76, 116-120. 41) Goldstein, J.C., and Brown, M.S. (1974) J. BioI. Chern. 249, 153- 162 42) Anderson, R.G.W., Vasile, E., Mello., R.I., Brown, M.S., and Goldstein, J.L. (1978) Cell 15, 919-927. 43) Willingham, M.W., Keen, J.C., and Pastan, I.H. (1981) Exptl. Cell Research 132, 329-338. 44) Goldstein, B., and Wofsy, C. (1981) Cell Biophys. 3, 251-277. 45) Gorden, P., Carpentier, J., Cohen, S., and Orci, L. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 5025-5029. 46) Willingham, M.L., Maxfield, F.R., and Pastan, I.H. (1979) J. Cell BioI. 82, 614-625. 47) Maxfield, F.R., Schlessinger, J., Shectiter,Y , Pastan, I., and Willingham, M.C. (1978) Cell 14, 805-810. 48) Larkin, J.M., Brown, M.S., Goldstein, J.L., and Anderson, R.G. (1983) Cell 33, 273-285. 49) McKanna, J.A., Haigler, H.J., and Cohen, S. (1979) Proc. Natl. Acad. Sci. U.S.A. 75, 5689-5693. 50) Bretscher, M.S., Thompson, J.N., and Pearse, B.M.F. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 4156-4159. 51) Payne, G.S., and Schekman, R. (1985) Science 23, 1009. 52) Anderson, R.G., Brown, M.S., and Goldstein, J.L. (1981) J. Cell BioI. 88, 441-452. 53) Wiley, S. (1988) 1. Cell BioI. 107, 801-810. 26 54) Carpentier, J., Gorden, P., Anderson, R.G., Goldstein, J.L., Brown, M.S., Cohen S., and Orci, L. (1982) J. Cell BioI. 95, 73-77. 55) Van Belzen, N., Rijken, P.I., Hage, W.I., DeLaat, S.W., Verkleij, A.I., and Boonstra, J. (1988) J. Cell BioI. 134,413-420. 56) Haigler, H.T., McKanna, J.A., and Cohen, S. (1979) J. Cell BioI. 81, 382-395. 57) Haigler, H., Ash, J.F., Singer, S.J., and Cohen, S. (1978) Cell BioI. 75, 3317-3321. 58) Bretscher, M.S. (1989) EMBO 8, 1341-1346. 59) Wiley, H.S. (1985) Curro Top. Mern. and Trans. 24, 369-411. 60) Carpenter, G., Lernbach, K.J., Morrison, M.M., and Cohen, S. (1975) J. BioI. Chern. 250, 4297-4304. 61) Wiley, H.S., and Cunningham, D.D. (1982) J. BioI. Chern. 257, 4222-4229. 62) Wiley, S., and Cunningham, D.D. (1981) Cell 25, 433-440. 63) Davis, R.I., VanDriel, I.R., Russell, D.W., Brown, M.S., and Goldstein, J.L. (1987) J. BioI. Chern. 262, 4075-4082. 64) Lazarovits, J., and Roth, M. (1988) Cell 53, 743-752. 65) McGraw, T.E., Pytowski, B., Arzt, J., and Ferrone C. (1991) J. Cell BioI. 112, 853-861. 66) Jing, S., Spencer, K.M., Hopkins, C., and Trowbridge I.S. (1990) J. Cell BioI. 110, 283-294. 67) Breitfeld, D.D., Cassanova, J.E., McKinnon, W.C., and Mostov, K.E. (1990) J. BioI. Chern. 265, 13750-13757. 68) Lobel, P., Fujirnuto, K., Ye, R.D., Griffiths, G., and Kornfeld, S. (1989) Cell 57, 787-796. 69) Wiley, H., S. (1988) J. Cell BioI. 107, 810-810. 70) Hari, J., and Roth R.A. 91987) J. Cell BioI. 262, 15341-15344. 71) McClan, D.A., Maegawa, H., Lee, J., Pull, TJ., Ulrich, A, and Tolefsky, S.M. (1987) J. BioI. Chern. 262, 14663-14671. 72) Russell, D.S., Gherzi, R.A., Johnson, E.L., Chou, C.K., Rosen, O.M. (1987) J. BioI. Chern. 262, 11833-11840. 27 73) Glenney Jr., J. R., Chen, W. S., Lazar, C. S., Walton, G. M., Zokas, L. M., Rosenfeld, M. G., and Gill, G. N. (1988) Cell. 52, 675- 684. 74) Chen, W.S., Lazar, C.S., Peoine, M., Tsien R.Y., Gill, G.N. and Rosenfeld M.G. (1987) Nature 328, 820-823. 75) Pool, R.R., Maurey., K.M., and Storrie, B. (1983) Cell BioI. Int. Repts. 7, 361-367. 76) Quintart, J., Courtoy, PJ., and Baudhuin, P. (1984) J. Cell BioI. 948, 877-884. 77) Storrie, B., Pool, R.R., Sachdeva, M., Maurey, K.M., and Oliver, C. (1984) J. Cell BioI. 948, 108-115. 78) Casey, K.A., Maurey, K.M., and Storrie, B. (1986) J. Cell Sci. 83, 119-113. 79) Courtroy, PJ., Quinzrt, J., and Baudhuin, P., (1984) J. Cell BioI. 98, 870-876. 80) Ajioka, R.S., and Kaplan, J. (1987) J. Cell BioI. 104,77-85. 81) Ajioka, R.S., and Kaplan J. (1986) Proc. Nati. Acad. Sci. U.S.A. 83, 6445-6449 82) De Duve, C., De Barsy, T., Poole, B., Trouel, A., Tukens, P., and Van Hood, F. (1974) Biochern. Pharmacol. 23, 2495-2527. 83) Hare, J.F. (1988) J. BioI. Chern. 263, 8759-8764. 28 84) Wiley, H.S., VanVostrand, W., McKinley, D.N., and Cunningham, D.D. (1985) J. BioI. Chern. 260, 5290-5295. 85) Geuze, H.1., Slot, J. W., Strouss,G.J., Lodish,H.F., and Schwartz,A.L. (1983). Cell 32,277-287. 86) Linderman, J.L., and Lauffenburger, D.A. (1988). J. Theor. BioI. 132, 203-245. 87) Griffiths, G., Black, R., and Marsh, M. (1989). J. Cell BioI. 109, 2703-2720. 88) Basu,S.K., Goldstein, J.L., Anderson, R.G., and Brown, M.S. (1981). Cell 24, 493-502. 89) Marshall, S., and Olefsky, J.M., (1983).1. Cell. Phys. 117, 295-303. 90) Levy, J.R., and Olefsky, J.M. (1988). J. BioI. Chern. 263, 6101- 6108. 91) Levy, J.R., Ullrich, A., and Olefsky, J.M. (1988). J. Clin. Invest. 81,1370-1377. 92) Ebner, R., and Derynck,R. (1991). Cell Regulation 2, 599-612. 93) Opresko,L., Wiley, H.S. and Wallace, R.A. (1980). Cell 22, 47-57. 94) Townsend, R.R., Wall, D.A., Hubard, A.L., and lee, Y.C. (1984). Proc. Natl. Acad. Sci. U.S.A. 81, 466-470. 95) Wandinger-Ness, A., Bennett, M.K., Antony, C., and Simons,K. (1990). J. Cell BioI. 111,987-1000. 96) Bartles,J.R., Feracci, H.M., Steiger,B., and Hubbard,A.L., (1987). J. Cell BioI. 105, 1241-1251. 97) Hunziker,W., and Mellman, I. (1989). J. Cell BioI. 109, 3291-3302. 98) Casanova,J.E., Breitfeld, P.P., Ross, S.A., and Mostov, K.E. (1990). Science 24,742-745. 99) Davis, C.G., van Driel,I.R., Russell, D.W., Brown, M.S., and Goldstein, J.L. (1987). J. BioI. Chern. 262,4075-4082. 29 100) Hunziker, W., Harter, C., Matter, K., and Mellman, I. (1991). Cell 66, 907-920. 101) Carpenter,G. and Cohen, S., (1976). J. Cell PhysioI. 88: 227-238. 102) Shechter, Y., Hernandez, L., and Herschman, H.R., (1978). J. BioI. Chern. 253: 3970-3977. 103) Knauer, D., Wiley, H.S., and Cunningham, D., (1984). J. BioI. Chern. 259: 5623-5631. 104) Gill, G., Bertics, P.and Santon, J., (1987). Molec. and Cell. Endrocrin. 51: 169-186. 105) Santon, J.B., Cronin, M.T., and MacLeod, C.L .. (1986). Cancer Res. 46: 4701-4705. 106) Wells, A., Welsh, J. B., Lazar, C. S., Wiley, H. S., Gill, G. N., and Rosenfeld, M. G.(1990). Science 247. 962-964 107) Carpenter,G., Cohen, S., (1976). J. Cell BioI. 71: 159-71. 108). Kahn,C., and Baird, K., (1978). J. BioI. Chern. 253: 4900-06. 109) Ascoli,M., and Puett,D., (1978). J.BioI. Chern. 253: 4892-99. 110) Chen, W. S., Lazar, C. S., Lund, K. A., Welsh, J. B., Chang, C. P., Walton, G. M., Der, C. J., Wiley, H. S., Gill, G. N., and Rosenfeld, M. G. (1989). Cell. 59: 33-43. 111) Glenney Jr., J. R., Chen, W. S., Lazar, C. S., Walton, G. M., Zokas, L. M., Rosenfeld, M. G., and Gill, G. N. (1988). Cell.52: 675-684. 112) Honegger, A. M., Dull, T. J., Felder, S., Van Obberghen, E., Bellot, F., Szapary, D., Schmidt, A., Ullrich, A., and Schlessinger, J. (1987). Cell. 51: 199-209 CHAPTER 2 THE ROLE OF LIGAND-INDUCED INTERNALIZATION IN DOWNREGULA TION OF THE EPIDERMAL GROWTH FACTOR RECEPTOR 31 Summary Occupancy-induced downregulation of cell surface EGF-Rs attenuates signal transduction. To define mechanisms through which downregulation of this class of growth factor receptors occurs, we have investigated the relative role of ligand-induced internalization in this process. It has been found that occupied, kinase-active EGF-Rs were internalized through a high affinity endocytic system at rates up to 10- fold faster than empty receptors. In contrast, full length EGF-Rs lacking tyrosine kinase activity underwent internalization at a rate independent of occupancy. This "kinase-independent" internalization rate appeared to reflect constitutive receptor internalization because it was similar to the internalization rate of both receptors lacking a cytoplasmic domain and of antibodies bound to empty receptors. All receptors that displayed ligand­induced internalization also underwent downregulation, indicating that the proximal cause of downregulation is occupancy-induced endocytosis. Tyrosine kinase activity appears to greatly enhances this process by stabilizing receptor association with the endocytic apparatus. The dependence of downregulation on ligand-induced internalization was further explored by examining the effects of the tumor promoter 12-0-tetradecanoyol-13-acetate (TPA) on on ligand­induced internalization in wild type and mutant EGF-Rs that contained an alanine substitution at position 654 (Ala654). Activation of protein kinase C with TPA prevented ligand-induced internalization of Thr654 32 receptors. TP A did not inhibit transferrin receptor internalization or constitutive EGF-R internalization, suggesting that protein kinase C activation inhibits only ligand-induced endocytosis. Inhibition of induced internalization by TPA required threonine at a.a. 654. Full-length Ala654 EGF-Rs were significantly resistant to TPA inhibition of ligand­induced internalization. The dominant regulatory effect of protein kinase C on the EGF-R appears to be mediated through phosphorylation at Thr654, disrupting the active receptor conformation required for kinase activity and ligand-induced internalization and thus preventing downregulation of the receptor. Introduction The cell surface is a dynamic structure in which specific components are continually added and removed. Among the most extensively regulated cell surface components are receptors. Receptors constitute the primary means by which cells perceive their environment. Although significant progress has been made in defining the mechanisms that regulate the internalization and sorting of receptors that bind metabolically important ligands such as transferrin (TO and low density lipoprotein (LDL), the mechanisms involved in downregulation of signal transducing receptors with intrinsic tyrosine kinase activity remain an important unsolved question in cell biology. The Tf and LDL receptors, whose primary function is bulk transport of nutritional molecules into the cell, cluster about coated pits 33 which constitute approximately 2% of the cell surface.(1,2) Internalization and trafficking of Tf and LDL receptors is independent of ligand binding. Clustering in coated pits, receptor internalization and subsequent recycling are observed in both the presence and absence of ligand.(1,3-6) Because the behavior of both occupied and unoccupied receptors is similar, ligand binding does not normally alter surface receptor number or intracellular distribution. In the case of LDL receptors, ligand dissociates in the acidic environment of the endosomes and is targeted to lysosomes while empty receptors recycle back to the cell surface(7). In the case of Tf receptors, ligand remains with the receptor and both recycle back to the cell surface.(8) In contrast to nutrient receptors, signaling receptors undergo ligand-induced conformational changes that alter their endogenous enzymatic activity and distribution. Empty EGF-Rs are randomly distributed at the cell surface and upon binding ligand they cluster in coated pits and are intemalized.(9,lO) Ligand binding also leads to activation of receptor tyrosine kinase activity and a decrease in surface receptor number (downregulation). Downregulation of EGF-Rs attenuates signal transduction and requires receptor kinase activity, but the mechanistic basis of the kinase requirement is controversial.(11-14) It has been proposed that in the case of full length EGF-R, activation of its intrinsic tyrosine kinase activity leads to high affinity binding to coated pits.(12,13,15) The resulting increase in internalization rate shifts receptors to an intracellular compartment which is then targeted to lysosomes.(16,17) However, other investigators have reported that 34 ligand-induced internalization of the EGF-R is independent of tyrosine kinase activity.(14,18-I9) Distinguishing between these alternate possibilities is important because little is known regarding the mechanisms of ligand-induced endocytosis or the processes that regulate receptor downregulation. In this chapter I detail experiments examining the relationship between ligand-induced internalization and downregulation of the EGF-R. I have also examined the influence of receptor tyrosine kinase activity on this process. Using labeled monoclonal antibodies to follow unoccupied receptors, I found that empty receptors undergo endocytosis at the same rate as receptors lacking the cytoplasmic domain, indicative of random entrapment by the endocytic apparatus. Occupancy of EGF-Rs lacking kinase activity did not change their internalization rate. However, occupancy of kinase-active receptors increased their internalization rate up to IO-fold. I conclude that ligand-induced EGF-R downregulation requires alterations in the receptors endocytic rate. Results Constitutive and Induced Internalization Rates of the EGF Receptor To determine the basal rate at which unoccupied EGF-Rs are internalized, two approaches were used. In the first approach, an EGF-R mutant that lacked a cytoplasmic domain was constructed because information that specifies high efficiency internalization for other 35 receptors is located in this region.(20-22) The internalization rate of this "intracellular-sequence-minus" (IS-) receptor should reflect random receptor entrapment by either smooth or coated pits. In the second approach, a monoclonal antibody (mAb 528) was used as a marker to follow empty receptors. This antibody does not activate the receptor and cannot bind to occupied receptors.(23) This allowed use of a double label protocol to simultaneously observe both occupied and unoccupied receptors. The IS- EGF-R truncation mutant (c'647) which retains only a two amino acid C' terminus past the transmembrane domain was prepared and evaluated in the ability of this receptor to both downregulate and to internalize EGF. As shown in Fig. 2.1, the IS- EGF-R was internalized at a low, but significant rate. EGF did not induce downregulation of this mutant receptor Also examined were the kinetics of both internalization and downregulation of full length kinase-active and kinase-inactive EGF-Rs under the same conditions used to evaluate the IS- construction. Kinase activity was required for both efficient internalization and downregulation (Fig. 2.1). The average internalization rate of the kinase-active receptor (0.32 min-I) was 8-fold higher than the M721 kinase-inactive receptor. Significantly, the internalization rate of EGF bound to full length kinase-inactive receptors was indistinguishable from that observed with the IS- receptor. 36 FIG. 2.1. Internalization and Downregulation of EGF Receptors. Cells expressed the c'647 1S- truncation (top panel), M721 kinase-inactive (middle panel) and wild type kinase-active (lower panel) EGF receptors. Left panels: Cells were incubated simultaneously with 0.8 nM 1311_EGF (-e-) and 1 nM 1251 labeled 528 monoclonal antibody (-0-). The specific internalization rate of the ligand was determined over a 5 min time period. All data are on the same scale and are presented as internalization plots. Right panels: Cells were treated with either 10 nM 528 monoclonal antibody (-0-) or 16 nM 1251_EGF (-e-) for the indicated times. Cells were then rapidly cooled and brought to equilibrium with their respective ligand. The number of surface receptors was determined by either flow cytometry for the monoclonal antibody or surface stripping followed by counting for EGF. 37 Internalization Down-regulation 30 • c'647(IS-) ~. • • • -100 20 ~ - !- 80 - !- 60 10 - !- 40 0 i ----Cf.::::::::::i: A.. _ Q"-::::::- ---- T I I 20 • Q) l§ "- :::I fI) (5 20 ~ ~ "0 cco:: g 10 i .~ ca c:: "- ~ .5 0 • M721 (Kin-) o--.!.. .. 0 0 • - 0 "'i .. .. .. - Q- 0 ____ 0 ---- 1'\.-0--- I , I 100 0~ ~ ~ 80 fI) c:: ~ £ 60 § "0 40 0- ~ <n 20 Wild Type (Kin+) ./ ~. P-o 0 - 0 -0- / - - .. ........... I- -...............- -8. 8 .. I- ~ o· "-,,8_0---°- ----0----- J I J 1 T T I 20 10 o 100 80 60 40 20 o 25 50 75 100 0 30 60 90 JSurface (% of total) Time (min) 38 The specific internalization rates of anti-EGF-R antibody bound to - wild-type, kinase-inactive and IS receptors were all similar (Fig. 2.1). These rates (0.03-0.05 min-I) were also similar to those displayed by EGF - bound to both kinase-inactive and IS EGF-Rs. Concentrations of antibody ranging from 0.1 to 12 nM did not affect internalization rates in cells expressing wild-type EGF-Rs (0.03 min-I). In addition, these concentrations of antibody did not induce receptor downregulation (Fig. 2.1), in agreement with previous studies using this antibody.(24) There was no significant difference (p>0.05) between the internalization rate of EGF bound to either the full length kinase-inactive or IS - receptors. In addition, there was no difference between the rate of antibody internalization displayed by kinase-active, kinase-inactive or IS EGF-Rs. The only significant difference was between the internalization rate displayed by kinase-active receptors binding EGF and all other receptors (p<O.Ol). These results indicate that the kinase-inactive receptors, IS- receptors and the unoccupied receptors all are entering the cell via constitutive internalization. To rule out the possibility that there were clonal differences in internalization rates of cells independent of the transfected EGF-R, the internalization rate of the transferrin receptor was determined in these cells. There was no significant difference in the internalization rate of the transferrin receptor in any of the cells tested (Fig. 2.2) indicating that the differences in the internalization ratewere due to the different 8000 .....------------------ - ..-, ~ 6000 >< (/) (l,) ::J U (l,) ~ 4000 --- ~ "0 (l,) .!::::! ~ 2000 '- -(l,) c: • WT Kinase + o M721 Kin- • N 2 Kin + o MN 2Kin- O~--------~----------~~----~~ a 2000 4000 6000 8000 10000 12000 Integral surface binding (molecule x min x cell-1 ) FIG. 2.2. Transferrin Internalization in B82 cells Expressing Human EGF Receptors. B82 cells expressing various constructs of the human EGF receptor were incubated with 1.7 nM 125I-Tffor 1-10 min. At 1 min intexvals, cell samples were rapidly shifted to O°C and the amount of radioactivity associated with the surface or inside of the cells was determined. The data are presented as internalization plots, in which the slope at any point is equal to the specific internalization rate of occupied receptors at that time. 39 40 constructs of the EGF-R in those cells. Only the kinase active construct that is capable of undergoing efficient ligand-induced internalization was also capable of downregulating the receptor in response to ligand. These results indicate that the proximal cause of downregulation is occupancy-induced endocytosis. TP A Inhibition of Ligand-Induced EGF Receptor Internalization The effect of TPA on EGF-R internalization was investigated. Cells that express wild-type EGF-Rs (Thr654) were treated for 10 min at 37°C with TPA and then incubated with 1251-EGF for 1-5 min. The specific internalization rate (ke) of the EGF-R was then determined. Fig. 2.3 shows that treatment with TP A decreased the value of ke from 0.31 min-1 to 0.03 min-I, indicating that TPA strongly inhibits ligand-induced internalization of EGF-Rs. The value of ke in the presence of TPA was 0.03 min-I, which is the same as constitutive EGF-R internalization observed with the kinase inactive receptors, unoccupied receptors and the IS- receptors. To ensure that inhibition of ligand-induced EGF-R internalization by TP A is not unique to B82 cells expressing transfected receptors, I examined both human fibroblasts and A431 cells. As shown in Fig. 2.4A, TP A reduced the specific intenlalization rate of EG F in each of these 41 2.5 ~ ("(') 2.0 I 0 ~ ~ ~ I .....-! 1.5 .....-! ~ U ~ 0 ~:r::J$'..) 1.0 / u ~ .....-! 0 's-' /0 ~ 0.5 .'"'-0 r:J'..) ~ _. -. o .---.- 0.0 .--- 0 2 4 6 8 10 J Surface (n10lecules x min x cell -Ix 1 0 -~ FIG. 2.3. TPA Inhibition of Ligand-Induced EGF Receptor Internalization. B82 cells expressing human EGF-R were incubated for 10 min at 37°C with 100 nM TP A (0) or control buffer (.). The cells were then incubated with 0.17 nM 125I-EGF for 1-5 min. At 1 min intervals, cell samples were rapidly shifted to O°C and the amount of radioactivity associated with the surface or inside of the cells was determined. The data are presented as internalization plots, in which the slope at any point is equal to the specific internalization rate of occupied receptors at that time. cell types. To determine \vhether the inhibition of internalization was specific to the EGF-R, the effect of TPA on transferrin receptor internalization was also measured. Endocytosis of the transferrin receptor is independent of receptor occupancy and occurs exclusively by a constitutive process.(3-7) In contrast to its inhibitory effect on EGF-R internalization, TPA increased the rate of transferrin receptor internalization in all cell types examined (Fig. 2.4.B). These results indicate that TPA selecti vely inhibits ligand-induced internalization of the EGF-R. Ala654 Receptors are Resistant to TPA Inhibition of Lif:and-induced Internalization 42 To determine the role of Thr654 phosphorylation in the inhibition of EGF-R internalization, the effect of TPA on internalization of Ala654 receptors was measured. As shown in Fig. 2.5, EGF·dependent endocytosis of Ala654 receptors was resistant to inhibition by TPA. Although TPA reduced the specific internalization rate of these receptors from 0.44 min-1 to 0.23 min-1 (Fig. 2.5), it failed to decrease ke to the low values typical of constitutive endocytosis (0.03 min-I) as with Thr654 receptors. Although they are resistant to complete inhibition of induced internalization by TPA, full-length Ala654 EGF-Rs do retain partial sensitivity. .91 ~ so:: 0.40 '.;:1 ~ 0.20 ~ -E ........ u ~........ u ~ 0.10 CI'l 0.00 A. EGF B .Transferrin Control II TPA B82 HF A431 882 HF A431 Cell Type 1.00 til toe:) 0.80 .~.... . ~ o.... .. ~ [ 0.60 ...... N ~. o t:S 1i1 tit 0.40'[ t:S 0.20 0.00 FIG. 2.4. Effect of TP A on the Rates of EGF and Transferrin Receptor Internalization. B82 cells expressing human EGF-Rs (B82), normal human fibroblasts (HF), and A431 cells (A431) were treated with 100 nM TPA ( • ) or control buffer ( D) for 10 min. The cells were then incubated with 0.17 nM 125I-EGF or 1 nM 125I-Tffor 1-5 min and the value ofke was determined by the internalization plot technique. 43 44 8 / r--.. /0 ("I") I 6 . 0 0 .,......; to< / ....... I ~ ~ Q) u to< 4 . r.I':J 0 ./ Q) ~~u';j / ./ 0 s ---- Q) '"0 2 . /.0 / .~ r.I':J ~ /1 -~~ - - - - - - 0 I I . I I I . 0 5 10 15 20 fSurface (molecules x min x cell -~ 10 -~ FIG. 2.5. Effect of TPA on Ala654 EGF Receptor Internalization. Cells expressing Ala654 EGF-Rs were treated for 10 min without ( 0 ) or with ( • ) 100 nM TP A. The cells were then exposed to 0.17 nM 125I-EGF for 1-5 min and the binding data converted to internalization plots. The value of ke is 0.44 min-1 in the absence of TP A and 0.23 min-1 in the presence of TP A. For comparison, the dashed line represents the slope of the internalization plot from Thr654 EGF-Rs in the presence of TPA where ke = 0.03 min-I. 45 TPA Prevents Ligand-induced Downregulation The hypothesis is that downregulation of the EGF-R results from induced internalization followed by receptor degradation.(6,9) To investigate the effect of TPA on receptor downregulation, cells expressing Thr654 or Ala654 EGF-Rs were treated with EGF in the absence and presence of TPA. Residual cell surface EGF-Rs were then measured by 125I-EGF binding. As shown in Fig. 2.6.A, B82 cells downregulate full-length Thr654 EGF-Rs to approximately 30% the control level within 1 hr of exposure to EGF. TP A alone induced a 20% reduction in the number of surface receptors but prevented ligand­induced downregulation of Thr654 EGF-Rs (Fig. 2.6.A). EGF also reduced the surface density of Ala654 receptors to approximately 30%, indicating that this substitution does not prevent receptor downregulation (Fig. 2.6.B). In contrast to its inhibitory effect on Thr654 receptor downregulation, TP A did not affect the ability of EGF to stimulate Ala654 receptor downregulation (Fig. 2.6.B). These results were confirmed by measuring surface receptor number by flow cytometry with an anti-EGF-R monoclonal antibody and by immunoprecipitation of 12sI-labeled surface receptors (data not shown). Thus, decreased ligand binding reflects a loss of receptor mass from the cell surface. Discussion A basic question regarding the diverse classes of membrane receptors that possess intrinsic tyrosine kinase activity is the molecular 120 A. WT (1;654) ,....-... --t g 100~ ~q ~ "\ 8 ~,~. ~ \ ~ '-' 80 \~. ~ Q----[] ~ o ~ ~ 60 ~ I ~ 40 20 ~o ~O i '\ '~O • o 15 30 45 60 0 15 30 45 60 time (min) 46 FIG. 2.6. Effect of TPA on EGF-induced Receptor Downregulation. B82 cells expressing human EGF-Rs were treated for 10 min without ( 0 ) or with ( • ) TP A prior to exposure to EGF at 37°C for the indicated times. Bound EGF was removed by exposure to acetic acid and residual EGF-Rs measured by incubating with 125I-EGF. A. Thr654 EGF-Rs. Control cells treated with TPA only ( D). B. Ala654 EGF-Rs. 47 mechanisms by which they are downregulated. Although it has been firmly established that the intrinsic tyrosine kinase activity of the EGF-R is required for biological activity, it has been less clear whether this enzymatic function is required for ligand-induced internalization. (12- 14,19,27-28) The current study provides several independent lines of evidence that intrinsic tyrosine kinase activity is required for rapid endocytosis of the EGF-R. There are three possible fates of all cell surface receptors with respect to coated pit internalization. Receptors can be specifically included in coated pits, they can be excluded from those structures or they can be neither included nor excluded. In the last case, the receptors should be internalized at a rate that reflects random capture by invaginating endocytic structures. This rate can be estimated by considering the fastest reported internalization rates of receptors. If a receptor is 100% captured by coated pits, then its specific internalization rate will equal that of the coated pit itself.(29) Carbohydrate-binding receptors have among the fastest internalization rates, usually ranging between 1.2-1.3 min-t,with the fastest reported rate being 4.1 min- 1.(30,31) Because coated pits comprise about 2% of the cell surface, random entrapment by coated pits should yield internalization rates of between 0.03-0.08 min-I. If random capture by smooth pits is included, the estimate is even higher. Recently, specific internalization rates of Tf receptors lacking either a cytoplasmic tail or a specific internalization sequence have been estimated, yielding values between 0.04-0.06 min- 48 1.(32,21) Similarly, replacing the cytoplasmic tail of the avian asialoglycoprotein receptor with unrelated sequences from Xenopus globin produced receptors with internalization rates between 0.02-0.06 min- I.(21) Finally, all "internalization defective" LDL receptors are internalized at a rate between 0.02-0.03 min-I.(20) Because these mutant LDL receptors are highly mobile in the plasma membrane, but are unable to cluster in coated pits, their internalization rate should accurately reflect random entrapment by coated pits or other endocytic structures.(3,29,33) All these data suggest that the specific internalization rate of randomly entrapped receptors will fall somewhere between 0.02-0.06 min-I. This is precisely the range of values we observed for kinase-inactive receptors (-0.03 min-I), unoccupied receptors and receptors lacking a cytoplasmic domain. The specific internalization rate of the ligand-bound, kinase-active -1 receptors averaged 0.32 min ,indicating that 32% of the occupied receptors were internalized per minute. In contrast, the internalization rate for ligand-occupied, kinase-inactive receptors was 0.03 min-I. The low internalization rate of kinase-inactive receptors was also shared by - EGF-Rs lacking a cytoplasmic domain (IS) and by unoccupied wild type receptors as measured by antibody internalization. Therefore, the large differences in endocytic behavior between kinase-active and kinase­inactive EGF-Rs appear to be explained by the ability of kinase-active EGF-Rs to bind with high affinity to coated pits or other components of the endocytic apparatus. All receptors that displayed a ligand-induced 49 increase in endocytic rates appear able to downregulate. Together, these data lead to the following model. In the absence of tyrosine kinase activity (empty receptors or occupied receptors modified by site-directed mutagenesis), EGF-Rs are internalized by random entrapment in endocytic structures. Ligand binding leads to a conformation change that allows interaction with coated pit components. Because ligand binding activates intrinsic protein tyrosine kinase activity, phosphorylation of some component by the EG F-R could stabilize this interaction, leading to the observed high affinity binding to the endocytic apparatus. Receptors modified by site-directed mutagenesis could bind either more tightly or less tightly to coated pits, depending on the structural consequence of the modification. However, "ligand-induced" internalization and subsequent receptor downregulation can occur only when receptor affinity for endocytic structures is significantly increased by occupancy. Differences in internalization rates between occupied and empty receptors correlate well with downregulation. Mutations in the EGF-R that impair occupancy-induced internalization rates impair occupancy­induced downregulation. Receptor mutations that enhance internalization rates also enhance downregulation.(12) Phosphorylation of the EGF-R at Thr654 by protein kinase C simultaneously blocks ligand-induced internalization and downregulation. In contrast, the data indicate that TPA did not impair constitutive endocytosis of EGF-Rs. The effects of protein kinase C on EGF-R activity are thus specific for ligand-induced 50 processes. Also, in contrast to Thr654 EGF-Rs, mutant Ala654 EGF-Rs were resistant to the inhibitory effects of TPA on ligand-induced endocytosis and these receptors were able to effectively downregulate. Together, all these data indicate that the proximal cause of EGF-induced receptor downregulation is occupancy-induced endocytosis. Materials and Methods General Mouse EGF was purified from submaxillary glands.(34) EGF, human, iron-loaded diferric Tf (Calbiochem-Behring Corp.) and monoclonal antihuman EGF-R antibody 528 IgG (23) were iodinated with either 1311 or 1251 (Amersham) using Iodo-Beads (Pierce) according to the manufacturers recommendations and free iodine separated from the radio labeled ligands by dialysis or by passing the mixture over a 0.8 x 20 cnl column of Sephadex G-10 equilibrated with PBS. The specific activity of 125I-Iabelled EGF was generally between 600 and 1,800 125 125 cpm/fmol, I-labelled Tf was between 780 and 3,100 cpm/fmol and 1- labeled 528 monoclonal was between 1,300 and 1,900 cpm/fmol. The 131 I-EGF was between 240 and 430 cpm/fmol. 12-0- tetradecanoylphorbol-13-acetate (TP A) (Sigma) was stored as a stock solution in DMSO or ethanol. Control experiments were performed using solvent alone. 51 Cell Culture B82 mouse L cells, which contain no endogenous EGF-Rs, and B82 cells transfected with normal (WT) or mutated (M721, c'647) human EGF-Rs were generated as previously described and were a gift from Dr. Gordon Gill.(12) A modified dihydrofolate reductase gene was the selectable marker for all transfections. B82 cells were grown in Dulbecco's modified Eagle's medium (DME, Flow Laboratories) containing dialyzed 10% calf serum (HyClone). Five J.!M methotrexate was added to the medium for those cells transfected with human EGF-R. A431 cells were obtained from Dr. Harry Haigler (University of California, Irvine) and grown in D ME containing 100/0 calf selum. Human foreskin fibroblasts were prepared and cultured as described.(35,36) Binding Studies Cells grown to confluence in 35 mm dishes were switched from growth medium to serum free DME containing 20 mM HEPES (pH 7.4) and 0.1 % BSA and no bicarbonate (D/H/B) 18 hrs before experiments. Binding experiments were initiated by changing to D/H/B containing the indicated concentrations of labelled ligand. The addition of ligand and the rinsing of cells were done with a semiautomatic apparatus.(35) The temperature limits for binding experiments were 35.5-37°C, and the temporal resolution was within 10 sec. Binding was terminated by rapidly rinsing six times with 2 mls ice-cold WHIPS buffer (20 mM 52 HEPES, pH 7.4,130 mM NaCI, 5 mM KCI, 0.5 mM MgCI2, 1 mM CaCI2, 1 mg/ml poly-vinylpyrrolidone). The relative amounts of ligand associated with the surface and interior of the cells was determined by o acid-stripping at 0 C using 50mM glycine-HCI, 100mM NaCI, 2mg/ml PVP, 2M Urea, pH 3.0. Stripping efficiencies were determined in parallel and were generally 98%. Nonspecific binding was determined in the presence of at least 200-fold molar excess unlabeled ligand in the case of Tf, or by measuring binding to B82 cells that lack EGF-Rs in the case of labeled EGF and 528 monoclonal antibody. Nonspecific binding was generally less than 5% of total binding. Single label experiments using 125I-Iabeled 528 IgG in the absence and presence of EGF established that EGF did not affect the specific internalization rate of the antibody. Cell nurnber was determined with a Coulter Counter. Downregulation To evaluate the ability of 528 monoclonal IgG to downregulate surface receptor expression, cells were incubated with 13 nM 528 IgG in standard binding medium at 37 0 C. At appropriate times, cells were removed from the plates by scraping and dispersed by trituration. After 6 centrifugation, aliquots of approximately 10 cells were resuspended in 50 J.11 of 528 monoclonal IgG at a concentration of 80J.1g/ml. After 30 min on ice, cells were washed twice with PBS and then FITC-conjugated anti-mouse IgG (50 III of 20 Ilg/ml) was added for another 30 min incubation on ice followed by washing with PBS and fixing in 1 % 53 glutaraldehyde in PBS. Five thousand cells per sample were analyzed using a Becton-Dickinson fluorescence-activated cell sorter with FACScan software. Mean fluorescence channels as the measurement of 4 fluorescence intensity were converted to a linear scale between 1 and 10 and reported as relative fluorescence units. Background fluorescence (receptor negative cells stained as described above) was subtracted from the relative fluorescence units. EGF-induced downregulation was also evaluated by incubating cells either with or without 50 nM 125I_EGF for the times indicated in the figure legends. Cells were then rapidly rinsed five times with ice-cold WHIPS solution and brought to equilibrium with 50 nM 125I_EGF 3-9 hrs.). Cells were again rinsed and the amount of surface-associated ligand determined by acid stripping. For studies using TP A cells expressing normal or mutant EGF-Rs were plated at 2 x 105 cells per well in 12-well plates. Twenty-four hrs later, the cells were treated with 50 nM TP A for 8 min at room temperature. The medium was then removed and the cells were washed once with binding buffer. The cells were then incubated at 37°C in binding buffer containing 50 nM EGF. At the indicated times, the cells were washed once with binding buffer and twice with 2.5 mM KCI, 135 mM NaCI, and 50 mM acetic acid at room temperature to remove surface-bound ligand.(37) The cells were then washed twice in cold binding buffer and incubated with 0.5 nM 1251 -EGF for 4 hr at 4°C. The cells were then washed twice, solubilized with 0.1 N NaOH/1 % sodium dodecyl sulfate, and counted in a gamma counter. Each point represents the mean of triplicate wells. The procedure to measure downregulation was verified by flow cytometry and Western blotting. References 1) Kaplan, J. (1981) Science 212, 14-20. 54 2) Anderson, R. G., Brown, M. S., and Goldstein, J. L. (1977) Cell 10, 351-364. 3) Beisiegel, U., Schneider, W. J., Goldstein, J. L., Anderson, R. G. W., and Brown, M. S. (1981) J. BioI. Chern. 256, 11923-11931. 4) Watts, C. (1985) J. Cell BioI. 100, 633-637. 5) Stein, B. S., and Sussman, H. H. (1986) J. BioI. Chern. 261, 10319-10331. 6) Ajioka, R. S., and Kaplan, J. (1986) Proc. Nati. Acad. Sci. U. S. A. 83, 6445-6449. 7) Brown, M. S., Anderson, R. G. W., and Goldstein, J. L. (1983) Cell 32, 663 -667. 8) Klausner, R. D., Ashwell, G., van-Renswoude, J., Harford, J. B., and Bridges, K. R. (1983) Proc. Nati. Acad. Sci. U. S. A. 80, 2263-2266. 9) Haigler, H. T., McKanna, J. A., and Cohen, S. (1979) J. Cell BioI. 81, 382-395. 10) Willingham, M. C., Haigler, H. T., Fitzgerald, D. J., Gallo, M. G., Rutherford, A. V., and Pastan, 1. H. (1983) Exp. Cell Res. 146, 163-175. 11) Wells, A., Welsh, J. B., Lazar, C. S., Wiley, H. S., Gill, G. N., and Rosenfeld, M. G. (1990) Science 247, 962-964. 55 12) Chen, W. S., Lazar, C. S., Lund, K. A., Welsh, J. B., Chang, C. P., Walton, G. M., Der, C. J., Wiley, H. S., Gill, G. N., and Rosenfeld, M. G. (1989) Cell 59, 33-43. 13) Glenney Jr., J. R., Chen, W. S., Lazar, C. S., Walton, G. M., Zokas, L. M., Rosenfeld, M. G., and Gill, G. N. (1988) Cell. 52, 675-684. 14) Honegger, A. M., Dull, T. J., Felder, S., Van Obberghen, E., Bellot, F., Szapary, D., Schmidt, A., Ullrich, A., and Schlessinger, J. (1987) Cell 51, 199-209. 15) Lund, K. A., Opresko, L. K., Starbuck, C., Walsh, B. J., and Wiley, H. S. (1990) J. BioI. Chern. 265, 15713-15723. 16) Stoscheck, C. M., and Carpenter, G. (1984) J. Cell BioI. 98, 1048- 1053. 17) Wiley, H. S. (1985) Curro Tops. Menlbr. Trans. 24, 369-412. 18) Honegger, A. M., Schmidt, A., Ullrich, A., and Schlessinger, J. (1990) J. Cell. BioI. 110, 1541-1548. 19) Felder, S., Miller, K., Moehren, G., Ullrich, A., Schlessinger, J., and Hopkins, C. R. (1990) Cell 61, 623-634. 20) Davis, C. G., van-Driel, I. R., Russell, D. W., Brown, M. S., and Goldstein, J. L. (1987) J. BioI. Chern. 262, 4075-4082. 21) Jing, S., Spencer, T., Miller, K., Hopkins, C., and Trowbridge, I. S. (1990) J. Cell BioI. 110, 283-294. 22) Verrey, F., Gilbert, T., Mellow, T., Proulx, G., and Drickamer, K. (1990) Cell Regulation 1, 471-486. 23) Gill, G. N., Kawamoto, T., Cochet, C., Le, A., Sato, J. D., Masui, H., McLeod, C., and Mendelsohn, J. (1984) J. BioI. Chern. 259, 7755-7760. 24) Kawamoto, T., Sato, J. D., Le, A., Polikoff, J., Sato, G. H., and Mendelsohn, J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 1337- 1341. 56 25) Carpenter, G. and Cohen, S. (1976) J. Cell BioI. 71, 159-171. 26) Stoscheck, C. M. and Carpenter, G. (1984) J. Cell BioI. 98, 1048- 1053. 27) Honegger, A. M., Szapary, D., Schmidt, A., Lyall, R., Van­Obberghen, E., Dull, T. J., lTllrich, A., and Schlessinger, J. (1987) Mol. Cell. BioI. 7, 4568-4571. 28) Chen, W. S., Lazar, C. S., Poenie, M., Tsien, R. Y., Gill, G. N., and Rosenfeld, M. G. (1987) Nature 328, 820-823. 29) Goldstein, B., Griego, R., and Wofsy, C. (1984) Biophys. J. 46, 573-58. 30) Magnusson, S., and Berg, T. (1989) Biochem. J. 257, 651-656. 31) Ward, D. M., and Kaplan, J. (1990) Biochem. J. 270, 369-374. 32) McGraw, T. E., and Maxfield, F. R. (1990) Cell Regulation 1, 369- 377. 33) Barak, L. S., and Webb, W. W. (1982) J. Cell BioI. 95, 846-852. 34) Savage, R. C., and Cohen, S. (1972) J. BioI. Chern. 247, 7609- 7611. 35) Wiley, H. S. and Cunningham, D. D. (1982) J. BioI. Chern. 257, 4222-4229. 36) Wiley, H. S. and Cunningham, D. D. (1981) Cell 25, 433-440. 37) Haigler, H. T., Maxfield, F. R., Willingham, M. C. and Pastan, I. (1980) J. BioI. Chern. 255, 1239-1241. CHAPTER 3 THE ROLE OF TYROSINE KINASE ACTIVITY IN RECYCLING AND COMPARTMENTALIZATION OF THE EPIDERMAL GROWTH FACTOR RECEPTOR 58 Summary The intrinsic tyrosine kinase activity of the epidermal growth factor receptor (EGF-R) is required for signal attenuation of the receptor through downregulation. Activation of the kinase activity leads to induced internalization of the receptor. This increased internalization rate is proposed to lead to a shift in the pool of receptors into the cell resulting in downregulation. However the mechanistic basis of the kinase requirement remains controversial as other investigators have reported that induced internalization of the EGF-R is independent of kinase activity and that downregulation is mediated through a kinase dependent inhibition of the recycling of the receptor. To further define the mechanism by which downregulation of the EGF-R occurs I have investigated the role of kinase activity in the recycling of the EGF-R. Transferrin and EGF internalized from either full length kinase active or full length kinase inactive receptors was efficiently recycled in these cells. The tl/2 of ligand return for the transferrin was approximately 5 min. In the average of five experiments the 0/2 of EGF loss was 14 +/- 2 and 16 +/- 7 min for full length kinase active and kinase inactive receptors respectively. These differences were not statistically significant (p > 0.05). Although the t1/2 of ligand loss of EGF was 3-fold slower than transferrin, EGF internalized from either full length kinase active or inactive receptors was colocalized within endosomes containing transferrin. Recycling was equally efficient in both full length kinase 59 active and inactive receptors and both receptors recycled using the same pathway as the transferrin receptor. Receptor tyrosine kinase activity is required for the downregulation of the EGF-R receptor and for the ligand-induced internalization of the receptor. However, recycling of the receptor is independent of the tyrosine kinase activity of the EGF-R. Introduction The binding of growth factors to a family of receptors that possess intrinsic tyrosine kinase activity elicits a wide variety of responses. Among these responses are the phosphorylation of intracellular substrates, increases in intracellular calcium, men1brane translocation, gene induction, and cell division. (1-5) How phosphorylation causes these changes remains unknown, but the kinase activity of the EGF-R is essential. Cells that have been transfected with the gene for the EGF-R become responsive to EGF unless the kinase activity of the receptor is abrogated. ( 6) Furthermore cells that have been transfected with a kinase active EGF-R, but are unable to regulate the receptor at the level of downregulation, display a transformed phenotype.(7) The cellular events occurring in response to EGF-R binding have been studied in great detail, yet little is known regarding the regulation of the activity of the EGF-R. Lack of regulatory control over the receptor can have dire consequences as is seen in the overexpression of the EGF-R in various tumors.(8,9) The EGF-R is regulated at the level of receptor number, receptor activity, substrate availability and receptor distribution within the cell. Regulation of EGF-R number can also occur at the level 60 of transcription. Abrogation of transcriptional regulation by chromosomal translocations can lead to overexpression of the receptor and a transformed phenotype in those cells.(9) Regulation of EGF-R activity, both positively and negatively, has been shown to occur via phosphorylation of the EGF-R itself.(10-12) Variability of the substrates for the kinase activity of the EGF-R has been observed in cells of different tissues. The availability of different substrates for the receptor has also been demonstrated to playa role in receptor activity.(13) Regulation of receptor distribution within the cell is the least understood type of receptor regulation. Regulation of receptor distribution within the cell involves the interaction of two processes: those that regulate the receptors internalization into the cell and those that target the internalized receptor either to the lysosomes or for recycling back to the cell surface.( 14) Alterations in either of these processes could have profound effects on the ability of the cell to attenuate the response elicited by the binding of EGF as well as alter the ability of the EGF-R to interact with key intracellular substrates for second messenger generation.(13-15) Most receptors are continuously internalized and recycled back to the cell surface. Insulin and transferrin receptors are recycled extensively, whereas the recycling of the EGF-R is dependent on cell type.(16-21) In the previous chapter I have shown that downregulation of the EGF-R requires ligand-induced internalization of the receptor that is dependent on the intrinsic tyrosine kinase activity of the receptor. 61 The resulting increase in internalization rate shifts receptors to an intracellular compartment which is then targeted to lysosomes.(14,18) However, other investigators have reported that ligand-induced internalization of the EGF-R is independent of tyrosine kinase activity and that downregulation is due to a kinase-mediated inhibition of EGF-R recycling. (22-24) A third possibility is that receptor kinase activity is involved in both endocytosis and postendocytic compartmentation. Distinguishing between these alternate possibilities is important because little is known regarding the mechanisms that regulate postendocytic sorting and targeting. The method used for determining the internalization rate (ke) of the receptors in Chapter 3 assumes that there is no appreciable recycling of the receptor during the time course used. Large amounts of recycling of the receptor during the 5 min time course of internalization would lower the apparent amount of internalized ligand and thus lower the apparent ke.(25,26) Therefore if the kinase inactive receptors were recycling at a significantly faster rate than the kinase active receptors the internalization rate would appear lower. This also could explain the differences in the ability of these receptors to downregulate. Therefore, in this chapter I have examined the influence of receptor kinase activity on recycling and intracellular compartmentalization of the EGF -R. I have found that both kinase active and inactive receptors recycle efficiently in B82 cells transfected with the human EGF-R. There are no significant differences in the rates of recycling between these receptors. 62 Additionally, I have determined that, although the recycling of the EGF­R in cells transfected with both kinase inactive and active EGF-Rs was 3- fold slower than the rate of transferrin receptor recycling, both EGF-Rs and transferrin receptors could be colocalized within the same recycling endosomes. I conclude that ligand-induced EGF-R downregulation is not mediated through alterations in the recycling rate of the receptor. Results Evidence for Recycling of the EGF-R: In/Sur Ratios Distribution of receptors within the cell is regulated by the transit times through the different compartments in the cell. If the mean lifetime inside the cell is expressed as Ti and the mean life time at the surface of the cell is expressed as Ts, then at steady state the ratio of receptor distribution between the inside and cell surface (the In/Sur ratio) can be expressed as the following. In/Sur= Tiffs When the life time inside the cell is increased by treatment with agents to prevent degradation the predicted result would be a large increase in the In/Sur ratio. Initial data suggesting recycling of the EGF-R were obtained by examination of In/Sur plots of transfected receptors in B82 cells. Cells expressing full length kinase active and inactive receptors were incubated with 125I-EGF in the presence or absence of 15uM monensin to prevent degradation. It has been previously demonstrated that monensin will inhibit intracellular degradation of both EGF and its receptor.(18,27-28) Although monensin will also block the recycling of 63 receptors in which ligand dissociation is a requirement for sOlting such as the LDL receptor, it has little effect on the recycling of membrane lipid and a relatively minor effect on other receptor types such as insulin or Tf receptors.(17, 29-31) At the indicated times the amount of surface bound and intracellular ligand was determined. As can be seen in Fig. 3.1 the treatment with monensin had no effect on the In/Sur ratio in either the cells expressing full length kinase active or kinase inactive receptors. Similar results were obtained with leupeptin. To ascertain that monesin was not inhibiting the internalization of the receptor monensin's effect on the internalization rate of the receptor was determined. As Fig. 3.2 demonstrates monensin had no effect on the internalization rate of either kinase active or kinase inactive receptors. These experiments indicate that internalized EOF is being lost from the cells via a recycling pathway. Recycling of EOF is Independent of Receptor Tyrosine Kinase Activity The internalization plot method for determining receptor internalization rates assumes that there is no loss of ligand from within the cell during the time of measurement, an assumption that has been validated in other cell types.(19,25,32) To ensure that differences between the behavior of kinase-active and inactive receptors were not due to differences in their rate of recycling this parameter was directly measured. 8 • WT --0- WTmonensin 6 -D-- M721 ~.CC.I.J.J • M721 Monensin = 4 -rJ-J CI.J .I,I'.Q.. CIl ~= 2 o o 20 40 60 80 100 120 140 Time min. FIG. 3.1. Inside/Surface Ratios of EGF in WT and Kinase Negative B82 cells. 64 WT cells in the presence (0) or absence (.) of 15uM monensin were incubated with 1.7nM 125I-EGF at 370 C. At the indicated times the amount of surface ligand was determined by acid stripping followed by detergent solubilization to determine the amount of internalized ligand. Kinase negative cells in the presence (0 ) or absence (_) of 15uM monensin were incubated with 17nM 125I-EGF at 370 C. At the indicated tin1es the amount of surface ligand was determined by acid stripping followed by detergent solubilization to determine the amount of internalized ligand. 65 3000 -.------r-_----------. 2000 1000 o OWT ke = 0.23 II WT + Monensin ke = 0.21 o M721 ke = 0.04 • M721 + Monensin ke= 0.03 o 10000 20000 30000 40000 50000 fSurface Binding (Receptors /ceU minute) FIG. 3.2. Internalization of WT and Kinase Negative EGF Receptors at Steady State. WT (D ) and kinase negative (0 ) cells were pretreated with either 1.6nM EGF(D,.) or 1.6nM EGF and 15uM monensin (-,0 ) for 2 hrs. After the pretreatment cells were incubated with the same solutions containing 125I-EGF. At 1 min time points the cells were rapidly cooled and the surface bound ligand was determined by acid stripping and counting.and the internalized ligand was determined by detergent extraction and counting. The data are presented as an internalization plot. 66 To determine whether receptor-associated ligands were rapidly recycled, we examined the rate at which previously internalized transferrin (Tf) and EGF were returned to the medium. Because Tf rapidly recycles while attached to its receptor, its use as an internal control permitted direct comparison between cells expressing kinase­active and kinase-inactive EGF-Rs. EGF was labeled with 1311 and Tf was labeled with 1251. Cells expressing either kinase-active or kinase­inactive receptors were exposed to a mixture of both ligands for 2 min followed by a chase in medium containing a high concentration of unlabeled ligands to prevent radiolabeled ligand rebinding. As shown in Fig. 3.3B, the recycling and discharge of Tf was identical for cells expressing either kinase-active or kinase-inactive EGF-Rs. The tl/2 of ligand return in this experiment was approximately 5 min, which was slower than the rate of fluid phase diacytosis.( 49) In multiple experiments, the measured time scale of Tf recycling was between 5 and 12 min in B82 cells, which is typical for this receptor. (33,34) EGF was also rapidly lost from the cells, but at a slower rate than Tf. The loss of Tf could be detected within 6 min of the initial ligand exposure, whereas loss of EGF was detected only after 8 min In the experiment shown in Fig. 3.3A, EGF internalized by cells expressing kinase-active receptors was lost with a tl/2 of 12 min while cells expressing kinase-inactive receptors lost EGF with a tl/2 of 25 min. The average of five separate experiments showed a tl/2 of EGF loss of 14 ± 2 min and 16 + 7 min for full length kinase-active and inactive receptors, respectively. 100- 80 60 40 Q) "C "w c: c:u "Ex 20 c:u 100- :2 +-' 80 c: Q) ~ 60 Q) c.. . 40 ~?O~~o A 0,// - ~-O_ --.......... 0--'0--0 -0- . ............ ......... EGF Pulse • • I • • I • ~ . ................ .... I • • B Transferrin 6 12 Time (min) 18 67 24 Fig.3.3. Recycling of EG F and Transferrin in B82 Cells. WT (e,. ) and kinase negative (0,0 ) cells were incubated with a combination of 131I-EGF (o,e) and 125I-Tf(D,.) for 2 min and then chased for the indicated times with excess unlabeled ligand to prevent rebinding . The amount of ligand left inside the cells was then determined by acid stripping and detergent extraction. 68 These differences were not statistically significant (p>O.05). Therefore, intrinsic kinase activity does not appear to be a primary regulator of this process. Intracellular Compartmentation of EG F Relative to Transferrin Although recycling of EGF through the cell was qualitatively similar to that of Tf, it was unclear whether the two ligands were using the same intracellular route. To clarify this issue, cells were incubated 125 131 for either 5 or 25 min with a combination of I-Tf and I-EGF. Surface-associated ligand was removed at 00 C followed by cell homogenization and fractionation. The distribution of EGF and Tf across the endosomal region of the density gradients was then evaluated. As shown in Fig. 3.4, endosomes were distributed over a relatively symmetrical peak centered at a density between 1.12 and 1.14 glml whereas the plasma membrane was found in the more dense region of the gradient (1.15-1.16 g/ml). The lysosomal marker hexosaminidase was well separated from the endosomes and distributed as a bimodal peak at 1.16 glml and 1.21 g/ml. Internalized EGF cosedimented with the endosomal Tf after either a 5 min or 25 min incubation, indicating that the same recycling pathway was utilized by both ligands. There was no difference in the compartmentation of EGF in cells expressing either kinase-active FIG. 3.4. Colocalization of EGF and Transferrin in Recycling Endosomes. 69 A. Sedimentation of endosomal transferrin. Cells \vere incubated for either 5 (_ ) or 25 (al )min with 6.5nM 125I~Tf. Surface ligand was removed and cells were fractionated on 15~50% isopynic sucrose gradients. Parallel groups of cells were incubated at OOC with 1251_ WGA to label the plasma membrane (PM). Mitochrondrial and lysosomal activity are labeled Mit and Lys respectively. B.Sedimentation of endosomal EGF.Celis were incubated simultaneously with 6.5nM 125I-Tf and 2nM 131I-EGF for either 5 (_ ) or 25 ( .. )min. Surface ligand was removed and endosomes were fractionated as in panel A. Shown is the distribution of EGF. The EGF{ff ratio was constant across the gradient. C Same experiment as above panel except the 131I-EGF concentration was 20nM and the cells express kinase negative receptors. The EGF{ff ratio was constant across the gradient. Top Bottom I~ A PM Lys .- ('I') 2.0 I 0 ,..- x E a.. '~-+(-)-- 1.0 Transferrin I LO C\.I 0.0 6.0 B 4.0 .- ('I') I 0 2.0 EGF (WT) .,...- x E a.. -(-)- 0.0 LL CJ W I -r- .M,.. . 6.0 C 4.0 EGF (M721) 2.0 o.o~--~--~--~--~--~--~----~--~ o 10 20 Fraction No. 30 40 70 1.2 1.17 0 1.14 co ::::J (J) ;::::;.: '< 1.11 co- -.... 3 1 .08 .::; 1.05 71 or kinase-inactive EGF-Rs (Fig. 3.4), indicating that postendocytic compartmentation is independent of intrinsic receptor tyrosine kinase activity. Although the average density of EGF -containing endosomes shifted to a lower density after 25 min, the same was observed for internalized transferrin (Fig. 3.4). Additionally the 125I-EGF/131I-Tf ratios remained constant after either 5 or 25 min pulses with ligand in both cells expressing kinase active and inactive receptors further indicating that postendocytic compartmentation is independent of intrinsic receptor tyrosine kinase activity and that the EGF and the Tf were sharing the same endosomal compartment (Fig. 3.5). To determine whether both Tf and EGF were in the same endocytic vesicles, we utilized the horseradish peroxidase-diaminobenzidine (DAB) density shift technique. (35,36) This method is based on peroxidase­H20 2-catalyzed oxidation of DAB within vesicles. The dense polymer of DAB which forms within the vesicle lumen increases the buoyant density of the vesicle. Thus, peroxidase-containing vesicles can be separated from other vesicles by density gradient centrifugation. Conjugates of HRP and both EGF and Tf were used to place peroxidase activity within endosomes containing the respective receptors. As shown in Fig. 3.6A, 125 incubation of cells simultaneously with both HRP-Tf and 1-Tf resulted in a density shift of endosomal 12\_ Tf after DAB-H202 treatment. This density shift required the presence of HRP-Tf (Fig. 3.6B). When 125 separate plates of cells were incubated with either HRP-Tf or I-Tf and then mixed prior to homogenization, no density shift occurred FIG. 3.5. Colocalization of EGF and Transferrin in Recycling Endosomes. Mitochrondrial and lysosomal activity are labeled Mit and Lys respectively. 72 Top Panel. WT cells were incubated simultaneously with 6.5nM 125I_ Tf and 2nM 131I-EGF for either 5 (_) or 25 (_ )min. Surface ligand was removed and endosomes were fractionated on 15-50% isopynic sucrose gradients. Shown is the distribution of EGF. The EGFrrf ratio was constant across the gradient at both 5(c ) and 25( 0 ) minutes. Bottom Panel. Same experiment as above panel except the 131I-EGF concentration was 20nM and the cells express kinase negative receptors. The EGFfff ratio was constant across the gradient for both 5(c) and 25 (0) minute incubations. 73 6.0 4.0 I I 00 I I WT w.......... .. io-oo4 ;a< I tT1 a ""!1 E 'S 0..0.0 U "-" N -'.../l ..I. ., c.r. 0 UJ -I "-l M- 0 A:l o=-· 1 0 M721 Fraction No. 74 FIG. 3.6. HRP-transferrin Conjugates can Density Shift Internalized EGF in 882 cells. Cells were incubated with 50 nM 125 125 of HRP-Tf and either I-Tf (panels A and B) or I-EGF (panels C and D) for 60 min at O°C followed by 15 min at 37°C. Surface­associated ligand was removed prior to homogenization. (A) Density shift of 125I_Tf, Homogenates were treated either without (_) or with (CJ) H20 2 and DAB prior to sucrose gradient fractionation. The shifted material was found in the 600/0 sucrose cushion at the bottom of the gradient. The nonshiftable material at the top of the gradient is ligand released during cell homogenization. B. Density shift requires colocalization of HRP and ligand. Cells were either treated with 1251_ Tf I f · 125 alone (:::::::::M:::;:~) or pates 0 cells were mcubated separately with I-Tf and HRP-Tf prior to mixing and homogenization (_) and then treated with H20 2 and DAB prior to sucrose gradient fractionation. (C) HRP-Tf wI.l l shl' ft 125 1-EG F. C e II s expressI.n g k'I nase-.l nactl. ve M 721 E G F receptors were incubated with both 125 1-EGF and HRP-Tf. Homogenates were treated either without (-) or with (C:l) H20 2 and DAB prior to sucrose gradient fractionation. (D) Same as panel C, but cells were expressing wild type EGF receptors. top HRP-Tf + 1251_Tf bottom top HR P-Tf + 1251-EGF bottom 6.0j A ~-D\r1 +DAB I C M721 I ~4.0 "'. 4.0 3.0 2.0 --. I\) M (II bTE)"" (" 2.0 1.0 -C.m".) n ~O.O 0.0 "'0 3 ~ B 0, 0 6.0 )( ..!. WT -4 I.() N 24 0 ..- W 4.0 16 8 2.0 0 0.0 10 20 30 40 10 20 30 40 Fraction No. ....,J Ul 76 (Fig. 3.6B). In a similar manner, endosomes containing both HRP-EGF and 125I_EGF could be shifted to the dense region of the gradient by DAB-H20 2 treatment (data not shown). These data confirm that density shifting occurs only when ligand and HRP are located in the same vesicle.(36) To demonstrate the colocalization of Tf and EGF within endosomes, cells expressing either kinase-active or kinase-inactive EGF­Rs were incubated simultaneously with 125I_EGF and HRP-Tf for 60 min at O°C followed by a chase for 15 min at 37°C. As shown in Fig. 3.6C 125 and 3.6D, the HRP-Tf was able to shift all endosomally-Iocalized 1- EGF to the bottom of the gradient. Significantly, there was no difference in the ability of HRP-Tf to shift EGF internalized by either the kinase­active or kinase-inactive EGF-R. These data indicate that the EGF-R traverses the same recycling endosomal pathway in B82 cells as does the Tf receptor. In addition, the protein tyrosine kinase activity of the EGF­R does not appear to qualitatively dictate its postendocytic compartmentation. Discussion The classical pattern of epidermal growth factor receptor (EGF-R) trafficking is as follows. The EGF-R is randomly distributed on the surface of cells bearing the receptor. Upon binding of ligand to the receptor the receptors cluster in coated pits and undergo a ligand-induced rapid internalization. After internalization the receptor- ligand complex 77 passes through several prelysosomal con1partments before degradation in the lysosome. Regulation of the EGF-R at the level of downregulation serves to limit the number of receptors that are activated at the cell surface and thus attenuates the signal generated by the receptor. Alterations in the internalization or intracellular trafficking of the receptor could abrogate the ability of cells to downregulate the EGF-R. In the previous chapter I reported that the kinase inactive EGF-R was unable to undergo downregulation in response to ligand. This inability to downregulate correlated with the inability of the receptor to undergo ligand-induced internalization. Other investigators have postulated that tyrosine kinase activity is required for EGF-R downregulation due to a kinase-mediated block in receptor recycling rather than at the level of ligand-induced internalization.(22-24) However, I could find no evidence to support this claim. Careful, direct measurement of the rate and extent of EGF recycling in cells expressing either kinase-active or kinase-inactive receptors indicates that EGF recycling is very similar for both receptors. Further, cell fractionation and density shift colocalization experiments demonstrate no qualitative difference between the postendocytic compartmentation of kinase-active or kinase-inactive receptors. Interestingly, although the recycling of both kinase active and inactive receptors are 3-fold slower than that of the transferrin receptor, both kinase active and inactive receptors can be found within the same recycling endosomes as examined by cell fractionation and density shift colocalization experiments. 78 The postendocytic trafficking pattern of EGF and its receptor appears to be highly cell-type dependent. Although the EGF-R recycles efficiently in transfected B82 cells and other cell types, there appears to be little if any EGF-R recycling in human fibroblasts. (18-21) Recycling of both EGF and its receptor is most evident in transformed cells which express high numbers of receptors, such as A431 or B82 cells.( 46) This could be due to the saturation of sorting endosome components that mediate lysosomal targeting. Overexpression of proteins normally targeted to lysosomes has been suggested to saturate the sorting mechanism in the trans-Golgi complex leading to their appearance at the cell surface. (47) Differences in cell function could be responsible for differences in the ability of cells to sort materials to the lysosomes. For example, the ratio of diacytosis to lysosomal targeting of endosomal contents varies between the apical and basolateral surfaces of epithelial cells during transcytosis. (48) An appealing hypothesis is that cell transformation is facilitated by a reduced expression of proteins that specifically target tyrosine kinase receptors to lysosomes, because this would also reduce growth factor utilization. Further studies are needed to critically test this hypothesis. Nevertheless, tyrosine kinase activity per se is clearly not required for the abrogation of recycling of the EGF-R. Materials and Methods General Mouse EGF was purified from submaxillary glands.(37) EGF, and human, iron-loaded diferric Tf (Calbiochem-Behring Corp.) were 79 iodinated with either 1311 or 1251 (Amersham) using Iodo-Beads (Pierce) according to the manufacturers recommendations and free iodine separated from the radiolabeled ligands by dialysis or by passing the mixture over a 0.8 x 20 cm column of Sephadex G-I0 equilibrated with PBS. The specific activity of 125I-labelled EGF was generally between 125 600 and 1,800 cpm/fmol, and I-labelled Tf was between 780 and 3,100 131 cpm/fnloL The I-EGF was between 240 and 430 cpm/fmoL Cell Culture B82 mouse L cells, which contain no endogenous EGF-Rs, and B82 cells transfected with normal (WT) or mutated (M721, c'647) human EGF-Rs were generated as previously described.(38) A modified dihydrofolate reductase gene was the selectable marker for all transfections. B82 cells were grown in Dulbecco's modified Eagle's medium (DME, Flow Laboratories) containing dialyzed 10% calf serum (HyClone). Five f.1M methotrexate was added to the medium for those cells transfected with human EGF-R. Binding Studies Cells grown to confluence in 35 mm dishes were switched from growth medium to serum free DME containing 20 mM HEPES (pH 7.4) and 0.1 % BSA and no bicarbonate (DIHIB) 18 hrs before experiments. Binding experiments were initiated by changing to DIHIB containing the indicated concentrations of labelled ligand. The addition of ligand and 80 the rinsing of cells were done with a semiautomatic apparatus.(39) The temperature limits for binding experiments were 35.5-37°C, and the temporal resolution was within 10 sec. Binding was tenninated by rapidly rinsing six times with 2 mls ice-cold WHIPS buffer (20 mM HEPES, pH 7.4, 130 mM NaCI, 5 mM KCI, 0.5 mM MgCI2, 1 mM CaCI2, 1 mg/ml poly-vinylpyrrolidone). The relative amounts of ligand associated with the surface and interior of the cells were detennined by acid-stripping at OOC using 50mM glycine-HCI, 100mM NaCI, 2mg/ml PVP, 2M Urea, pH 3.0. Stripping efficiencies were determined in parallel and were generally 98%. Nonspecific binding was determined in the presence of at least 200-fold molar excess unlabeled ligand in the case of Tf, or by measuring binding to B82 cells that lack EGF-Rs. Nonspecific binding was generally less than 5% of total binding.Cell number was determined with a Coulter Counter. Gradient Fractionation of Cells Subconfluent monolayers of cells grown in 100 mm plates were incubated with radiolabeled ligand at the indicated times at 37°C. After rinsing, surface-associated ligand was removed with 100 mM NaCI, 50 mM Glycine, pH 3.0 for 2 min followed by rinsing with saline and 100 J.1M phenylarsine oxide for 10 min at O°C to render the cells fragile. Cells were removed from their plates by incubation with 600J.1g/ml trypsin at O°C followed by neutralization with 5-fold excess soybean trypsin inhibitor. Cells were pelleted and homogenized in 1 ml of 250 81 mM sucrose, 10 mM triethanolamine, 10 mM acetic acid, 1 mM EDT A, pH 7.4, by 8 passages through a 25 gauge needle. Cell breakage was generally 80-90% as assayed by microscopy. Nuclei were removed by centrifugation at 900 x g for 3 min Samples were loaded on linear 19- 40% (w/w) sucrose/PVP gradients and brought to isopynic equilibrium by centrifugation in an SW 40 rotor at 37,500 rpm for 12 hrs. at 4 °C.( 40) Gradients were pumped out of the top of the tubes using an ISCO model 185 density gradient fractionator at a rate of 1 nll/min. Fractions of 0.3 ml were either counted or evaluated for enzymatic . . 125 131 activity. Samples contaInIng both I and I were counted on a three channel Packard gamma spectrophotometer. All counts were corrected for channel spillover and counting efficiency. Sedimentation position of the plasma membrane was determined by binding of 125I_Iabeled wheat germ agglutinin or by surface iodination using sulfo-SHPP (Pierce Chemical Co) at O°C. Both methods yielded identical results. Endosomes 125 were identified by incubating cells to a steady state with 1-Tf followed by removal of the surface-associated ligand with glycine/HCI prior to cell fractionation. Lysosomes were identified by hexosaminidase activity and mitochondria by cytochrome-C oxidase activity .(41,42) Density profiles were determined by refractive index. Density Shifting of Endosomes Colocalization of different ligands within endosomes was established by the horseradish peroxidase (HRP)-diaminobenzidine (DAB) 82 density shift technique. (35,36) The conjugate between Tf and HRP (HRP-Tf) was a gift of Dr. Jerry Kaplan. A 1:1 conjugate of EGF and HRP (HRP-EGF) was prepared by the method of Nakane and Kawaoi. (43) The HRP was purified by CM-cellulose chromatography followed by conjugation to EGF.(44) The HRP-EGF was isolated by sequential chromatography on P-100 (Bio-Rad Laboratories) and CM-Cellulose using a 0.05-0.1 M sodium acetate gradient (pH 4.4). The peak eluting at 0.06 M acetate was confirmed to be a 1: 1 EGF:HRP conjugate by absorbance ratio at 280 and 400 nM as well as enzymatic activity and ability to compete with 125I_EGF for receptor binding. Cells in 100mm dishes were incubated with 50 nM of either HRP-Tf or HRP-EGF and 50 125 125 nM of either 1-EGF or 1-Tf for 60 nlin at O°C followed by a shift to 37°C for 15 min. Surface-associated ligand was renloved by acid stripping and the cells were homogenized using 10 passages through a ball-bearing homogenizer. (45) The postnuclear supernatants were treated with DAB and H20 2 and then fractionated by isopynic sucrose gradients as described above.(36) References 1) Hunter,T., Alexanser, C.B. and Cooper, J.A. (1985) Ciba. Found. Symp. 116, 118-204. 2) Moolenaar,W.H., Aerts, R.J., Tertoolen, L.G. and de Laat,S.W. (1986) J. BioI. Chern. 261, 279-284. 3) Gill, G.N., Bertics, PJ. and Stanton, J.B. (1987) Mol. Cell. Endocrinol. 51, 169-186. 83 4) Carpender, G. and Cohen S. (1979) Ann. Rev. Biochem. 48, 193- 216. 5) Wiley, H.S. and Kaplan, J. (1984) Proc. Nati. Acad. Sci. U.S.A. 81, 7456-7460. 6) Chen, W.S., Lazar, C.S., Poenie, M., Tsien, R.Y., Gill, G.N. and Rosenfeld,M.G. (1987) Nature 328, 820-823. 7) Wells, A., Welsh, J. B., Lazar, C. S., Wiley, H. S., Gill, G. N., and Rosenfeld, M. G. (1990). Science 247.962-964. 8) Santon, J.B., Cronin, M.T., and MacLeod, C.L. (1986). Cancer Res. 46: 4701-4705. 9) Libermann, T.A., Nusbaum, H.R., Razon, N., Kris, R., Las, I., Soreq, H., Whittle, N., Waterfield, M.D., Ullrich, A. and Schlessinger, J. (1985) Nature 313,144-147. 10) Gill, G.N., Chen, W.S., Lazar, C.S., Glenney Jr., J. R., Wiley, H.S., Ingraham, H.A. and Rosenfeld,M.G. (1988) Cold Spr. Symp. Quant. BioI. 53, 467-476. 11) Sibley,D.R., Benovic, J.L., Caron, M.G. and Lefkowitz, R.J. (1987) Cell 48, 913-922. 12) Davis, R.J. (1988) J. BioI. Chern. 263, 9462-9469. 13) Blay, J., Valentine-Braun, K.A., Northup, J.K. and Hollenberg, M.D. (1988) Biochem., J. 259, 577-583. 14) Wiley, H.S. (1985) Curro Tops. Membr. Trans. 24, 369-412. 15) Wiley, H.S. and Cunningham, D.D. (1981) Cell 25, 433-440. 16) Krupp, M.N. and Lane, M.D. (1982) J. BioI. Chern. (257,1372- 1377. 17) Huecksteadt, T., Olefsky, J.M., Brandenberg, D. and Heidenreich, K.A. (1986) J. BioI. Chern. 261, 8655-8659. 84 18) Stoscheck,C.M. and Carpenter, G. (1984) 1. Cell. BioI. 98, 1048- 1053. 19) Dunn, W.A. and Hubbard, A.L. 1. (1984) Cell. BioI. 98,2148-2159. 20) Kroc, M. and Magun, B.E. (1985) Proc. NatI. Acad. Sci. U.S.A. 82, 6172-6175. 21) Sorkin, A., Komilova, E., Teslenko, L., Sorokin, A. and Nikolsky, N. (1989) Biochim. Biophys. Acta. 1011, 88-96. 22) Honegger, A. M., Dull, T. 1., Felder, S., Van Obberghen, E., Bellot, F., Szapary, D., Schmidt, A., Ullrich, A., and Schlessinger, 1. (1987) Cell 51,199-209. 23) Honegger, A. M., Schmidt, A., Ullrich, A., and Schlessinger, 1. (1990) 1. Cell. BioI. 110, 1541-1548. 24) Felder, S., Miller, K., Moehren, G., Ullrich, A., Schlessinger, 1., and Hopkins, C. R. (1990) Cell 61, 623-634. 25) Wiley, H. S., and Cunningham, D. D. (1982) 1. BioI. Chern. 257, 4222-4229. 26) Waters, C. M., Oberg, K. C., Carpenter, G., and Overholser, K. A. (1990) Biochemistry 29, 3563-3569. 27) King, A. C. (1984) Biochem. Biophys. Res. Commun. 124, 585- 591. 28) Wiley, H. S., W. VanNostrand, D. N. McKinley, and Cunningham, D. D. (1985) 1. BioI. Chern. 260, 5290-5295. 29) Koval, M. and Pagano, R. E. (1989) 1. Cell BioI. 108,2169-2181. 30) Stein, B. S., and Sussman, H. H. (1986) 1. BioI. Chern. 261, 10319- 10331. 31) Basu, S. K., Goldstein, 1. L., Anderson, R. G. W., and Brown, M. S. (1981) Cell 24, 493-502. 32) Opresko, L. K., and Wiley, H. S. (1987) 1. BioI. Chern. 262, 4116- 4123. 85 33) McGraw, T. E., and Maxfield, F. R. (1990) Cell Regulation 1, 369- 377. 34) Jing, S., Spencer, T., Miller, K., Hopkins, C., and Trowbridge, I. S. (1990) J. Cell BioI. 110, 283-294. 35) Courtoy, P. J., Quintart, J., and Baudhuin, P. (1984) J. Cell BioI. 98, 870-876. 36) Ajioka, R. S., and Kaplan, J. (1987) J. Cell BioI. 104, 77-85. 37) Savage, R. C., and Cohen, S. (1972) J. BioI. Chern. 247, 7609- 7611. 38) Chen, W. S., Lazar, C. S., Lund, K. A., Welsh, J. B., Chang, C. P., Walton, G. M., Der, C. J., Wiley, H. S., Gill, G. N., and Rosenfeld, M. G. (1989) Cell 59, 33-43. 39) Wiley, H. S. and Cunningham, D. D. (1982) J. BioI. Chern. 257, 4222-4229. 40) Opresko, L. K., Wiley, H. S., and Wallace, R. A. (1980) Cell 22, 47-57. 41) Sottocasa, G. L., Kuylenstierna, B., Ernster, L., and Bergstrand, A. (1967) J. Cell BioI. 32, 415-437. 42) Horvat, A., Baxandall, J., and Touster, O. (1969) J. Cell BioI. 42, 469-479. 43) Nakane, P., and Kawaoi, A. (1974) J. Histochem. Cytochem. 22, 1084-1091. 44) Shannon, L. M., Kay, E., and Lew, J. Y. (1966) J. BioI. Chern. 241, 2166-2172. 45) Balch, W. E., and Rothman, J. E. (1985) Arch. Biochem. Biophys. 240, 413-425. 86 46) Sorkin, A., Krolenko, S., Kudrjavtceva, N., Lazebnik, J., Teslenko, L., Soderquist, A. M., and Nikolsky, N. (1991) J. Cell BioI. 112, 55-63. 47) Williams, M. A., and Fukuda, M. (1990) J. Cell BioI. Ill, 955- 966. 48) Bomsel, M., Prydz, K., Parton, R. G., Gruenberg, J., and Simons, K. (1989) J. Cell BioI. 109, 3243-3258. 49) Wiley, H.S., Herbst, J.1., Walsh, B.J., Lauffenburger,D.A., Rosenfeld, M.G. and Gill, G.N. (1991) J. BioI. Chern. 266, 11083- 11094. CHAPTER 4 SEQUENCES WITHIN THE EPIDERMAL GROWTH FACTOR RECEPTOR THAT DIRECT LYSOSOMAL TARGETING 88 Summary The mechanism of occupancy induced downregulation of the epidermal growth factor receptor (EGF-R) remains controversial. Previous chapters have focused on the internalization and recycling of kinase active and kinase inactive receptors. In this chapter the role of kinase activity and other sequences within the EGF-R in directing lysosomal targeting of the receptor for eventual degradation was examined. Whether degradation is necessary for downregulation of the receptor will also be examined. I determined that degradation is not necessary for downregulation of the EGF-R. Treatment of cells with monensin to prevent degradation had no effect on the ability of these cells to downregulate. Furthermore, cells expressing both kinase active and inactive receptors degraded EGF concordant with their relative extent of internalization. Utilization of an assay to simultaneously measure the extent of recycling and degradation revealed that kinase inactive and active receptors recycle and degrade proportionally to the same extent. These results were confirmed using sucrose gradients to measure the transfer of ligand to the lysosomal compartment. These gradients revealed that both kinase active and kinase inactive receptors were able to traffic ligand to the late endosomes/lysosomes. Monitoring of inside/surface ratios using either a monoclonal antibody or ligand revealed that occupied receptors are 89 retained within the cell much more efficiently than unoccupied receptors. These results indicate that the ability of the EGF-R to be retained within the cell is dependent upon ligand occupancy. Monitoring of the extent of recycling and degradation in cells expressing various constructs of the EGF-R revealed that transfer to lysosomes for degradation is independent of kinase activity and independent of the internalization domain of the receptor. Transfer to lysosomes was dependent on a domain of the receptor adjacent to the plasma membrane between amino acids 647 and 688 of the receptor. Cells expressing the receptor truncated 2 amino acids distal to the transmembrane region at a.a. 647 were unable to degrade EGF while cells expressing a receptor truncated at a.a. 688 were capable of degradation. The ability to transfer receptors away from the recycling pathway to the degradative pathway is a saturable process. In experiments monitoring the fractional recycling and degradation of EGF the larger the internal pool of occupied receptors the smaller the fraction of EGF that was degraded. I conclude that degradation is not necessary for ligand-induced downregulation of the EGF-R. Lysosomal sorting of the EGF-R is not dependent on kinase activity of the receptor or on the internalization domain of the receptor. Lysosomal sorting of the EGF-R is a saturable process dependent on sequences within the cytoplasmic tail of the EGF-R between a.a.647 and 688 of the receptor. 90 Introduction Mammalian cells internalize a wide variety of molecules through receptor mediated endocytosis. Following internalization these molecules can have widely different fates. Transferrin and the LDL receptor are extensively recycled whereas other molecules such as EGF are extensively degraded.(1-3) An early endosomal compartment called CURL (compartment for uncoupling of receptor and ligand) is the site at which sorting of receptors for either recycling or degradation occurs.( 4) Evidence for protein sorting signals comes from a variety of sources. One source of evidence comes from the study of polarized epithelium in hepatocytes. Hepatocytes are a polarized cell with distinct apical and basolateral plasma membrane domains. During membrane biogenesis all membrane proteins are shipped first to the basolateral domain. Apical proteins are then internalized and transcytosed to the apical surface.(5) This vectorial transport implies targeting sequences within the apical proteins to direct their transport and specialized proteins to recognize these sequences. Indeed differences in the composition of apical and basolateral transport vesicles have been reported.(6) Another model used for the study of protein sorting is the MDCK cell. Golgi to basolateral sorting in these cells has been found to involve cytoplasmic determinants in several proteins. The polymeric immunoglobulin receptor ( pIgR) requires a negative charge at position 664 in the cytoplasmic tail that allows entry into coated pits and transcytosis.(7,8) 91 Not all sorting signals are similar to those required for clustering in coated pit