Characterization of the movement of receptors and ligands through the endocytic apparatus in alveolar macrophages.

A number of studies have outlined general features of the endocytic pathway. Specific details are lacking regarding the factors that regulate the rates of movement of receptors and ligands within the endocytic pathway, as well as the organelles which constitute the pathway. These studies demonstrated that the rates of internalization of different receptor-ligand complexes, in a single cell type, are different, and that the specific internalization rate of a receptor-ligand complex was unaffected by either the degree of receptor occupancy or the simultaneous internalization of different receptor-ligand complexes in alveolar macrophages. Different receptor-ligand complexes were shown to be internalized into the same endosomes. After internalization, the degradation of different ligands was initiated at the same time, suggesting that ligands that move to the degradative compartment move at the same rate regardless of whether ligands were internalized by receptor-mediated or fluid-phase pinocytosis. Different receptors move back to the cell surface at the same rate, regardless of whether they were initially unoccupied or occupied. The fusion of sequentially internalized vesicles was examined in alveolar macrophages and HeLa cells. The results demonstrated that most ligands internalized at different times mixed within early endosomes, thus demonstrating fusion of sequentially internalized vesicles. The fusion of early endosomes appeared to involve a long lived fusion competent vesicle in which receptor and ligand sorting occurred. Transferrin molecules were shown to move into and out of this sorting compartment very rapidly such that sequentially internalized transferrin molecules did not mix in early endosomes. Additionally, sequentially internalized transferrin molecules did not mix in recycling endosomes. Ligands destined for the lysosome appeared to move rapidly into and out of fusion competent early endosomes. Within the late endocytic pathway sequentially internalized vesicles were capable of fusing prior to the lysosome. This later fusion compartment was defined as a late endosome based on the absence of transferrin-transferrin receptors and classic lysosomal markers, such as acid glycosidases. Ligand degradation occurred in this compartment, indicating the presence of proteases. The ability of late endosomes to fuse with demonstrated using lowered temperatures and/or a methodology that inhibited late endosome-lysosome fusion, the substitution of K+ for Na+ in incubation buffers. It was determined that the substitution of K+ for Na+ buffers prevented endosome-lysosome fusion in a number of different cell types. These studies also included an investigation of the mechanisms(s) by which iso-K+ solutions inhibited endosome movement. A number of potential agents or molecules were ruled out as being causal to the inhibition of endosome-lysosome fusion including: pH changes, alteration in microtubules, and increases in intracellular cyclic-AMP or Ca[2+]. Studies using ion substitution, or sucrose addition, identified changes in cell volume as being causal to derangements in late endosome movement.

CHARACfERIZA TION OF THE MOVEMENT OF RECEPTORS AND LIGANDS THROUGH THE ENDOCYTIC APPARATUS IN ALVEOLAR MACROPHAGES by Diane McVey Ward A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirement for the degree of Doctor of Philosophy Department of Pathology The University of Utah March 1991 CopyrightoDiane McVey Ward 1991 All Rights Reserved THE UNIVERSITY OF UTAH GRADUATE SCHOOL SUPERVISORY COMMITTEE APPROVAL of a dissertation submitted by Diane McVey Ward This dissertation has been read by each member of the following supervisory commi ttee and by majority vote has been found to be satisfactory. / ~i THE UNIVERSITY OF UTAH GRADUATE SCHOOL FIN AL READING APPROV AL To the Graduate Council of the University of Utah: I have read the dissertation of Diane McVe¥ Ward in its final fonn 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. Approved for the Major Department Approved for the Graduate Council B. Gale Dick Dean of The Graduate School ABSTRACf A number of studies have outlined general features of the endocytic pathway. Specific details are lacking regarding the factors that regulate the rates of movement of receptors and ligands within the endocytic pathway, as well as the organelles which constitute the pathway. These studies denl0nstrated that the rates of internalization of different receptor-ligand complexes, in a single cell type, are different, and that the specific internalization rate of a receptor-ligand complex was unaffected by either the degree of receptor occupancy or the simultaneous internalization of different receptor-ligand complexes in alveolar macrophages. Different receptor-ligand complexes were shown to be internalized into the same endosomes. After internalization, the degradation of different ligands was initiated at the same time, suggesting that ligands that move to the degradative compartment move at the same rate regardless of whether ligands were internalized by receptor-mediated or fluid­phase pinocytosis. The rate of recycling of receptors back to the cell surface was measured showing that different receptors move back to the cell surface at the same rate, regardless of whether they were initially unoccupied or occupied. The fusion of sequentially internalized vesicles was examined in alveolar macrophages and HeLa cells. The results demonstrated that most ligands internalized at different times mixed within early endosomes, thus demonstrating fusion of sequentially internalized vesicles. The fusion of early endosomes appeared to involve a long lived fusion competent vesicle in which receptor and ligand sorting occurred. Transferrin molecules were shown to move into and out of this sorting conlpartment very rapidly such that sequentially internalized transferrin molecules did not mix in early endosomes. Additionally, sequentially internalized transferrin molecules did not mix in recycling endosomes. Ligands destined for the lysosome appeared to move rapidly into and out of fusion competent early endosomes. Within the late endocytic pathway sequentially internalized vesicles were capable of fusing prior to the lysosome. This later fusion compartment was defined as a late endosome based on the absence of transferrin-transferrin receptors and classic lysosomal markers, such as acid glycosidases. Although classic lysosomal markers were missing, ligand degradation occurred in this compartment, indicating the presence of proteases. The ability of late endosomes to fuse was demonstrated using lowered temperatures and/or a methodology that inhibited late endosome-lysosome fusion, the substitution of K+ for Na+ in incubation buffers. It was determined that the substitution of K+ for Na+ buffers prevented endosome-lysosome fusion in a number of different cell types. These studies also included an investigation of the mechanism(s) by which iso-K+ solutions inhibited endosome movement. A number of potential agents or molecules were ruled out as being causal to the inhibition of endosome-lysosome fusion including: pH changes, alteration in microtubules, and increases in intracellular cyclic-AMP or Ca2+. Studies using ion substitution, or sucrose addition, identified changes in cell volume as being causal to derangements in late endosome movement. v To Scott TABLE OF CONTENTS ABSTRACf ..... . ACKNOWLEOOMENTS Chapter 1. INTRODUCTION Internalization . . 2. 3. 4. Early Endosomes ......... . Dissociation of Ligand-Receptor Complexes Recycling Vesicles . . . . The Late Endocytic Pathway . . . . . . References . . . . . . . . . . . . . THE RATE OF INTERNALIZATION OF DIFFERENT RECEPTOR-LIGAND COMPLEXES IN ALVEOLAR MACROPHAGES IS RECEPTOR SPECIFIC Introduction. . . . . Materials and Methods Results Discussion . . . . . References . . . . . . COHORT MOVEMENT OF DIFFERENT LIGANDS AND RECEPTORS IN THE INTRACELLULAR ENDOCYTIC PA TIIW A Y OF ALVEOLAR MACROPHAGES Experimental Procedures. Results Discussion . . . . . . References . . . . . . FUSION OF SEQUENTIALLY INTERNALIZED VESICLES IN ALVEOLAR MACROPHAGES Abstract . . . . . . . . . . . Materials and Methods Results ..... . Discussion . . . . . . IV ix . 4 .... 8 10 13 15 18 27 28 28 29 32 33 34 35 36 39 40 42 43 44 44 50 5. 6. 7. APPENDIX. References . . . . . . . . . . . . . . . . . . . . . 51 INHIBmON OF LATE ENDOSOME-LYSOSOME FUSION: MECHANISM BY WHICH ISOTONIC-K+ BUFFERS ALTER UGAND MOVEMENT . Materials and Methods Results ..... Discussion . . . Acknowlegdments References . . . PRELIMINARY EXPERIMENTS INVESTIGATING ENDOSOME FUSION IN HELA CELLS AND J774 CELLS: FUSION OF INTERNALIZED VESICLES WITH A PRE-EXISTING ORGANELLE. Abstract . . . . . . Introduction. . . . . . Materials and Methods Results .. Discussion . . . . . . References . . SUMMARY USE OF THE HORSERADISH PEROXIDASE/DIAMINO­BENZIDINE DENSITY SHIFT TECHNIQUE TO STUDY IN1RACELLULAR 53 55 55 60 61 61 63 64 64 66 67 82 84 86 VESICLE TRAFFICKING .............. 90 viii ACKNOWLEDGMENTS I want to thank Jerry Kaplan for the opportunity of working in his laboratory, for his undying support and advice, and especially for teaching me how to ask the right questions. I also wish to express my appreciation to David P. Hackenyos, Richard S. Ajioka, Sandra Davis-Kaplan, and Lisa Gren for their technical assistance. Chapter 2 (Ward, D.M. and Kaplan, J. 1990. The rate of internalization of different receptor-ligand complexes in alveolar macrophages is receptor specific. Biochem J. 270:369-374) is published with the permission of The Biochemical JournaL Chapter 3 (Ward, D.M., Ajioka, R. and Kaplan, J. 1989. Cohort movement of different ligands and receptors in the intracellular endocytic pathway of alveolar macrophages. 1. BioI. Chern. 264:8164-8170) is published with the pennission of the American Society of Biochemistry and Molecular Biology. Chapter 4 (Ward, D.M., Hackenyos, D.P. and Kaplan, J. 1990. Fusion of sequentially internalized vesicles in alveolarmacrophages. 1. Cell BioI. 110:1013 .. 1022) is published with the pernlission of Rockefeller Press. Chapter 5 (Ward, D.M., Hackenyos, D.P., Davis­Kaplan, S. and Kaplan, J. 1990. 1. Cell. Physio!. 145:522-530) is published with the pennission of Wiley-Liss Inc. CHAPTER 1 INTRODUCTION 2 Alveolar macrophages are highly specialized cells, one of whose major functions is to remove deleterious agents from biological fluids. These agents may include pathogenic bacteria, particulate debris, and a variety of biologically active molecules such as enzymes and toxins. The macrophage also plays a major role in the turnover of extracellular constituents including aged red cells, polymorphonuclear neutrophils, extracellular matrix and surfactant. To perform these functions macrophages internalize extracellular molecules. The molecules are then directed to intracellular compartments for degradation and/or utilization. While other cell types are capable of internalization, macrophages have a number of special features which make them highly efficient "scavengers. II Internalization may occur by fluid-phase pinocytosis, "cell drinking" (Lewis 1931), a constitutive, although regulated process. Additionally, particles, by binding to the cell surface as a consequence of nonspecific electrochemical forces may be internalized (Silverstein et al. 1977, Stossel 1974). Most interactions which result in the efficient internalization of particles/ligands, are mediated by specific molecules present on the plasma membrane (Goldstein et ale 1979, Anderson and Kaplan 1983). These receptors specifically recognize either conformational or structural features of molecules in the extracellular environment. Internalization can fulfill a number of functions. As described, internalization results in the clearance of potentially injurious molecules. Two examples are alpha-macroglobulin.protease (aM.P) and mannose terminal glycoproteins (MTG). Alpha M.P complexes are composed of the protease inhibitor aM bound to one of number of endoproteases. The endoprotease, while covalently bound to the aM, still retains enzymatic activity. Macrophage accumulation of these complexes results in the removal of these potentially injurious molecules. Among the ligands recognized by the MTG receptor are lysosomal enzymes. These enzymes are released as a result of cell death, but are still functional and may damage tissue if not removed. Many receptors, upon binding ligand, transmit signals such as the hydrolysis of phosphotidyl inositol lipids, or the generation of cyclic AMP. Internalization of either the receptor-ligand complex, or the signal transduction system, provides a means of terminating the signal. 3 Macrophages have an immense capacity to internalize molecules. Measurement of the volume of fluid internalized by pinocytosis indicate that macrophages are capable of internalizing a volume of extracellular fluid equivalent to their own volume every hour (Steinman et al. 1976). It has also been demonstrated that macrophages are capable of internalizing a quantity of particles whose surface area is equivalent to their own (Petty et al. 1981). The ability of macrophages to maintain a high rate of internalization is due to the fact that most internalized membrane components are not degraded, but are cycled back to the cell surface and thus reutilized (Petty et al 1981). Through selective membrane recycling, cells may regulate the quantity and types of membrane components exposed on the cell surface, thereby affecting their response to changes in their environment. Endocytosis, while demonstrated by most cells types, is important for macrophage function and macrophages are an excellent cell type in which to study endocytosis. Alveolar macrophages express a variety of different receptors (Kaplan and Nielson 1979a, 1979b, Stahl et aI. 1980, Buys et aI. 1984). These receptors provide markers that can be followed to address questions regarding the endocytic apparatus and intracellular trafficking. A number of macrophage receptors discussed in this dissertation are termed "scavenger" receptors, whose major role is to clear injurious ligands from biological fluids. These receptors bind ligand and the receptor-ligand complex is internalized. Within the cell the complex is dissociated and ligand is routed to the lysosome where it is degraded. The unoccupied receptor is recycled to the cell surface (Stahl et al. 1980, Buys et al. 1984). Examples of such receptors which are used in these studies are aM.P receptors, MTG, and the acetylated-Iow density lipoprotein (LDL) receptor described by Brown et al. (1980) which recognizes maleylated-bovine serum aIbumin (MAL-BSA). Macrophages also contain transferrin (Tf) receptors, a receptor whose function is to provide cells with an 4 essential metal. These receptors bind and internalize diferric transferrin (Tf(Feh). Within the cell iron in released, and the apoTf-Tf-receptor complex is recycled to the cell surface (VanRenswoude et al. 1982., Buys et al. 1987). While the endocytic pathway is known in some detail, substantial questions remain regarding the regulation of endocytic activities and intracellular traffic. The following paragraphs include a brief discussion of the known features regarding internalization and membrane trafficking and discuss some questions that remain to be answered. Internalization Receptor-ligand internalization has been heavily studied for two reasons. Internalization is the step that distinguishes surface receptors from internal receptors, and receptor-ligand complex internalization is relatively easy to follow. Measurements of receptor internalization are possible because ligands may be tagged, and simple procedures are available to separate free ligand from cell associated ligand. For example, some ligands bind to their receptors in a pH and divalent cation dependent manner. Chelation of divalent cations or reduction in pH results in the dissociation of surface bound ligand. Thus, internalized and surface bound ligand can be distinguished by placing cells in buffers of low pH containing divalent cation chelators (Haigler et al. 1983, Kaplan and Nielson 1979b, Wiley and Cunningham 1981, Wiley 1988). Protease treatment of cells at OOC have also been used to distinguish surface bound (protease susceptible) from internalized (protease resistant) ligand. Studies have shown that the rate of receptor-ligand complex internalization varies widely. Some membrane components exhibit a half time of internalization of less than a minute (Wiley 1985, Magnusson and Berg 1989), while others have a halftime of greater than 12 hours (Presky and Schonbrunn 1986). Most studies have measured the internalization rate of one type of receptor-ligand complex in one cell type. Few have examined the rates of internalization of different receptors in the same cell type. Those 5 studies which have focused on internalization rates have focused prin1arily on the internalization of hormone-receptor complexes. These studies demonstrate a wide variation in internalization rate depending on the particular hormone studied (Wiley and Cunningham 1982, Presky and Schonbrunn 1986). In some instances internalization of receptor­hormone complexes is associated with termination of a ligand-induced signal (Segaloff and Ascoli 1981). The variations in internalization rate may reveal more about signal transduction than they do about the endocytic machinery. While there is a suggestion that different receptors will exhibit different rates of internalization, formal studies have been lacking. Chapter 2 presents studies addressing this issue demonstrating that, in a single cell type, different receptor-ligand complexes are internalized at different rates. A number of factors influence internalization rates. Some receptors are internalized constitutively, that is the rate of internalization is independent of occupancy (Ajioka and Kaplan 1986). It is unclear what factors determine whether receptors are internalized constitutively or in a ligand dependent manner. In some cells, internalization of Tf receptors and EGF receptors is ligand dependent (Klausner et al. 1984). In other cell types internalization is partially or totally independent (Carpentier and Cohen 1976, Korc and Magsun 1985). Additionally, the addition of a hormone or pharmacologic agent may alter the ligand dependency (Buys et al. 1987, Opresko and Wiley 1987b, May et al. 1984). Given a deflned rate of internalization, the question have been raised as to whether the specific internalization rate is altered as a function of receptor occupancy. That is, does intraspeciflc competition exist for the internalization apparatus when the number of receptor-ligand complexes available for internalization increase? If so, one might expect to see a decrease in speciflc internalization rates. Kaplan and Keogh (1983) demonstrated that aM.T was internalized at a similar rate at either 10% or 80% surface occupancy. Ciechanover et al (1983) demonstrated that Tf receptors and asialoglycoprotein receptors 6 were internalized at rates that were independent of occupancy. Other studies have, however, demonstrated that the specific internalization rate of epidermal growth factor (EGF) receptors in A431 cells (Wiley 1988) and vitellogenin receptors in Xenopus oocytes (Opresko and Wiley 1987b) was affected by the number of occupied receptors. The rates of internalization decreased with increased receptor occupancy. The interpretation of this data was that a competition exists between receptors for binding to the internalization apparatus. In A431 cells the EGF receptor is expressed in extraordinary quantities, on the order of 2-5 x 106 receptors Icell. This is a 10-to 100-fold increase in receptor number beyond any measurement reported to date in macrophages (Stahl et al. 1980, Kaplan and Nielson 1979a,1979b, Buys et aI. 1984). Another example ofinternaIization rates being altered by the number of receptor-ligand complexes to internalize is the vitellogenin receptor in.Xenopus oocytes (Opresko and Wiley 1987a, 1987b). Another possible way in which the rates of internalization of receptor-ligand complexes may be affected is if there is interreceptor competition for the endocytic apparatus. Ciechanover et al. (1983) has demonstrated that the internalization of an asialoglycoprotein-asialoglycoprotein receptor complex is unaffected by the simultaneous internalization of Tf-Tf receptor complex. Buys and Kaplan (1987) have also demonstrated, in alveolar macrophages, that receptor-ligand complex internalization is unaffected by the uptake of zymosan, polystyrene beads, or IgG coated red blood cells. These results suggest that there is little competition among receptors for an internalization apparatus. The studies in Chapter 2 also demonstrate little competition among receptors for an internalization apparatus. It may be that the number of receptors in a given cell type are unable to saturate the system, or there may be multiple systems for internalization. Most receptor-mediated endocytosis occurs through coated pits (Goldstein et al. 1979, Anderson et aI. 1977, Willingham et aI. 1981, Wall et al. 1980, Carpentier et al. 1982, Willingham et al. 1979, Harding et al. 1983). These structures are apparent as 7 invaginations, or depressions in the plasma membrane in which the cytoplasmic surface is enriched with proteins termed clathrin and clathrin associated proteins (Roth and Porter 1964, Pearse 1976). Clathrin is composed of heavy and light chains both that are required to assemble into a triskelion structure, which then forms the clathrin "basket." In fibroblasts, the concentration of clathrin is such that about 2.0% of the cell surface is considered to be coated (Anderson et al. 1977). In macrophages, the percentage of surface area considered coated is much higher, 10-20% (Aggelar and Werb 1982). The exact role of clathrin is unclear. Initially, clathrin was considered necessary for all forms of endocytosis (Marsh and Helenius 1980, Goldstein et al. 1979, Gorden et aL 1978). It has become clear, however, that a number of types of endocytosis can occur in the absence of coated pits (Sandvig et aI. 1987, Racosin and Swanson 1989, Goldberg et aL 1987). It has been difficult, however, to determine the amount and types of endocytic activity mediated by coated pits or by noncoated membranes. The absolute amount of membrane internalized, as well as the number of coated pits, are cell type dependent and are affected by both honnonaI and environmental factors. Most receptors are internalized through coated pits, suggesting that the function of the coat may be to tether or capture receptors. Clathrin-associated proteins, termed adaptins, have been suggested to playa role in the binding of receptors to coated pits. Bainton et al. (1987) and Glickman et aI. (1989) have demonstrated, in vitro, the binding of adaptins to the cytoplasmic tails of receptors. These studies aIso demonstrated that different receptors can compete for binding to adaptins, suggesting that adaptins are the proteins that link receptors to coated pits. The linkage to coated pits has yet to be defined. Adaptins may recognize some structure or amino acid sequence of a receptor in the correct conformation. Site-specific mutagenesis of a number of receptors have demarcated structural features and, in some instances, amino acid residues necessary for internalization (Lehnnan et al. 1985, Davis et al. 1987 Lobel et aI. 1989, Mostov et aI. 1986, Prywes et aL 1986, McClain et al. 1987, Rothenberger 1987, Miettinen et aL 1989, Chen et aI. 1990, McGraw et al. 1990). 8 A consensus sequence or structure that governs internalization has not been established. Internalization of receptor-ligand complexes occurs through the clustering of these complexes in areas of the plasma membrane which subsequently are pinched off. How planar membranes pinch off to form vesicles is undefined. Morphologic studies indicate that shallow or flask shaped pits invaginate until they form vesicles (Anderson et aL 1977, Schwartz et al. 1982, Basu et al. 1981, Stahl et al. 1980). Studies have demonstrated that when coated pits are destroyed, by alterations in intracellular pH or ion content, internalization of receptor-ligand complexes is inhibited and clustered receptors become dispersed on the cell surface (Heuser and Anderson 1989). These results demonstrate the importance of the coated pit in the internalization of receptor-ligand complexes. Preclustering of receptors in coated pits may be a factor that results in internalization rate differences among receptors. In Chapter 2 the ability to reversibly alter membrane receptor topology is used to examine the effect of receptor clustering on the specific internalization rates of different receptor-ligand complexes. After internalization, there is a short period in which clathrin remains associated with the vesicle, now referred to as a coated vesicle. Clathrin rapidly dissociates from the internalized vesicle, and this dissociation appears to be mediated by an A TP-requiring enzyme (Schmid et al. 1985). After clathrin dissociates the internalized vesicle is now called an early endosome. Early Endosomes Little is known about the constituents of the early endocytic vesicle. One important activity demonstrated to be in endosomes is an A TP-driven H+ pump that acidifies the lumen of the vesicle (Forgac et al. 1983). This acidification is required for receptor-ligand complex dissociation (Basu et al. 1982, Robbins et al. 1983, Tietze et al. 1980) as well as the release of iron from Tf(Fe)2 (Klausner et al. 1984). Mutants in the H+ pump exist 9 (Robbins et al. 1983, Stone et al. 1987), and the endosomal H+ transport pump has been characterized and isolated (Stone et al. 1987, Xie et al. 1983). Studies have demonstrated that the pH of the early endocytic vesicle is approximately 6.5, a pH that is much higher than that of late endosomes (Fuch et al. 1989, Pearse 1987, Yamashiro et al. 1983). Recent studies have suggested a novel mechanism by which the pH of the early endosome is regulated. The pH appears to be determined by both the action of the H+ pump as well as by the activity of the Na+/K+ ATPase (Cain et al. 1989, Fuch et al. 1989). The activity of internalized Na + /K+ ATPase brings about the electrogenic movement of ions, resulting in the formation of a potential difference across the endosomal membrane, with the luminal surface being positively charged. This electrical gradient prevents the entry of H+, restricting luminal pH. When the Na+/K+ ATPase is removed, as would occur during the membrane sorting process, the potential difference dissipates allowing for an increase in H+ transport and a decrease in pH. Inhibition of the Na+/K+ATPase by ouabain, both in vitro and in vivo, results in an increased acidification of early endosomes. Several issues about early endosome functions and capabilities remain unclear. Many groups have demonstrated in vitro vesicle fusion of early endosomes (Braell 1987, Davey et al. 1985, Diaz et al. 1988, Gruenberg et al. 1989). These studies have been performed in a wide variety of cell types using receptor ligands, fluid-phase ligands and viral proteins. Additionally, the in vitro studies have begun to determine the biochemical requirements for fusion of these early endosomes. While these studies have described fusion of early endocytic vesicles, they have not provided much information on the endocytic pathway. Morphologic studies indicate that early endocytic vesicles disappear. Internalized ligand is now found in a larger structure in which receptor-ligand dissociation may occur. It is not clear how this "sorting" compartment is formed. Does this compartment result solely from the fusion of early endosomes, or is it the results of fusion of early endosomes with some preexisting structure (Helenius 1983, Roederer et al. 1987, Griffith 1989)? It is within this compartment that the fate of the ligand and/or ligand-receptor complex is 10 detennined, thereby constituting an important landmark in the endocytic pathway. Ajioka and Kaplan (1987) demonstrated that Tf internalized at different times remain segregated within the endocytic apparatus. These results suggest that early and recycling endosomes do not fuse and mix their contents. Salzman and Maxfield (1988, 1989), however, demonstrated endosome fusion in Chinese hampster ovary (CHO) cells using both Tf and a fluid-phase ligand. Further studies by the same group demonstrated fusion of sequentially internalized vesicles which contain Tf or LDL. Their studies indicated that while internalized LDL accumulated in a central compartment, Tf containing early endosomes fused with this "sorting" compartment but then rapidly exited. The differences between the studies of Ajioka and Kaplan (1987), and those of Salzman and Maxfield (1988, 1989) and Dunn et al. (1989) may be related to different cell types, ligands, or procedures used. Although the differences observed may be due to methodologies, the overall conclusion from the studies presented here, and those listed above, is a consensus view of the early endosomal pathway. Dissociation of Li ~and -Receptor Complexes It is clear that receptors and ligands may have different fates, some receptors and ligands being recycled and others being degraded. Some receptor-ligand complexes dissociate, the receptor is recycled and the ligand is sorted to a storage or degradative compartment. Other receptor-ligand complexes may be shuttled to the lysosome intact. Little is known about the regulation of movement of receptors and ligands and the factors that determine the fates of different receptors and ligands. As den10nstrated biochemically and morphologically, dissociation of receptor-ligand complexes appears to occurs within this sorting compartment. The separation of iron (Fe) from Tf has been demonstrated to occur in this compartment (Klausner et al. 1984). Morphological studies by Geuze and colleagues have visualized this organelle, tenned 11 CURL (Compartment of Uncoupling of Receptor- Ligands) as a pan shaped structure in which the recycling receptors are present in the pan handle and the dissociated ligand in the fluid-phase of the pan (Geuze et al. 1983, 1984). The surface area/volume relationship of this structure has been used to explain how receptors and ligands can have different fates. Pinching off of the receptor-rich "handle," as would occur for membrane destined to be recycl~ back to the surface, would trap little fluid and thus return only small amounts of nonreceptor bound ligand to the surface. Ligand, now present in the fluid-phase of the remaining organelle would be directed to the degradative compartment. It has been suggested that pH is the major factor affecting receptor-ligand dissociation. This conclusion has been based on studies that have used a variety of approaches including weak bases (Tietze et al. 1980), proton ionophores (Basu et al. 1981), endosomal pH mutants (Klausner et al. 1984, Stone et al. 1987) and genetically engineered receptors (Davis et al. 1987). It is clear that decreased pH is an important factor in regulating ligand-receptor affmity, however, studies indicate that other factors must also be involved. One factor that may playa role in receptor-ligand dissociation is the concentration of divalent ions, particularly the Ca2+concentration within the endosomal lumen. The relationship between pH and Ca2+ has been demonstrated in a study by Harford et al. (1984). These authors demonstrated that at a given pH, chelation of Ca2+ accelerated the dissociation of asialoglycoprotein-asialoglycoprotein receptor complexes. Dissociation of some ligand-receptor complex occurs independent of pH or divalent cations. For example, the dissociation of maleylated-bovine serum albumin or acetyl-low density lipoprotein (ligands that bind to the macrophage "scavenger receptor") does not appear to be affected by Ca2+ chelation or low pH (Brown et aI1980). Opresko and Wiley (1987 a) have shown that vitellogenin is dependent upon Ca2+ and pH for binding to its receptor, but chelation of Ca2+ to remove ligand is only 60% efficient and "acid stripping" was not effective. A non-physiological alkaline pH was required to induce receptor-ligand dissociation. Since both of these receptors are known to recycle, mechanisms other than 12 Ca2+ removal or lowered pH must exist to mediate ligand-receptor dissociation. The rate of ligand dissociation may be critical in determining receptor fate. The time, or "window," in which receptor-ligand complexes dissociate may be limited by the rate of vesicle movement. If ligand dissociation occurs before a receptor exits the early sorting compartment or CURL, then the receptor may be recycled. Conversely, if receptor-ligand dissociation is not accomplished in this time frame, a greater percentage of receptors will be shunted to the lysosome or back to the cell surface. A small percentage of ligand-receptor complexes may normally be returned to the cell surface, a process referred to as diacytosis or retroendocytosis (Bestennan et al. 1981, Auslinkas et al. 1981, Tietze et al. 1982, Greenspan and St. Clair 1984, Chang and Kullberg 1984, Marshall 1985, McKinley and Wiley 1988). It is not clear, however, whether this reflects the return of an unoccupied receptor and ligand that is free in the lumen of the endosome, or receptor-ligand complexes that simply have not dissociated as they passed through the CURL. Conditions which decrease ligand-receptor dissociation may tend to increase the amounts of receptors destined for degradation. This argument has been used to explain how the multivalency of Fc receptors in macrophages can determine whether the Fc receptor is recycled or degraded (Leinhard 1983, Mellman et al. 1983, Mellman and Plotner 1984, Ukkonen et al. 1986). Fc receptors bind and mediate the internalization of IgG. When IgG is internalized as a monomer, the Fc receptor recycles. Alternatively, when IgG is internalized as part of an antigen-antibody complex, the receptor is degraded. Formation of antigen-antibody complexes increases receptor-ligand affinity, and consequently reduces the rate of dissociation. The decrease in dissociation rate results in a greater proportion of internalized receptors being occupied and thus a greater proportion being sent to the lysosome. The above observations lead to a strong hypothesis as to how the fate of receptors and thus receptor concentration can be regulated, in part, by the rate of movement of molecules through the endocytic apparatus. 13 Receptors and ligands, in the sorting compartment, in most instances have a minimum of two fates. They can enter a population of recycling vesicles and be returned to the cell surface, or they can be targeted to the lysosome for degradation. [Other possibilities exist, depending upon the cell type, such as transcytosis that occurs in epithelial and endothelial cells (Simons and Fuller 1985)]. Below is a discussion of what is know~ about recycling and late endosomes and what has yet to be defined. Recyclin~ Vesicles Recycling endosomes are those vesicles that will fuse with the plasma membrane. Their identification has been based on their constituents. They may contain unoccupied or occupied receptors. Unoccupied receptors represent those receptors that have been recently vacated, and those receptors that may have been originally internalized unoccupied. Occupied receptors, on the other hand, may represent those complexes that do not dissociate in the sorting compartment, complexes that are unable to dissociate, or complexes which become "trapped" in the recycling vesicle prior to dissociation. The paradigm of an occupied recycling receptor is the Tf-Tf receptor complex which releases the iron bound to transferrin in the sorting organelle and the complex is then recycled to the surface (Van Renswoude et al. 1982). There is also some evidence that high density lipoprotein-receptor complexes recycle as a unit (Oram et al. 1983). The path or intracellular route taken by recycling vesicles is somewhat controversial, particularly the question of whether recycling receptors move through the Golgi apparatus. The Golgi apparatus is continually budding off vesicles that may be directed to the plasma membrane, packaged into secretory granUles, or directed to lysosomes (Griffith and Simons 1986, Dunphy and Rothman 1985, Farquhar 1985). These vesicles have been shown to contain newly synthesized receptors as well as receptors which bind lysosomal enzymes (VonFigura et al. 1986, Griffiths et al. 1988). There is some evidence that Tf receptors desialylated at the cell surface may be resialylated during recycling by moving 14 through the trans-Golgi (Snider and Rogers 1985). The amount of Tf receptors that are seen to move through the trans-Golgi and the kinetics of movement, however, do not correspond to the recycling rates measured for Tf receptors. Stoorvogel et al. (1988) demonstrated, using electron microscopy and biochemical techniques, that recycling Tf receptOrs are found in the trans-Golgi reticulum and not in the Golgi itself. These data would suggest that the majority of internalized receptors do not recycle through the Golgi apparatus. A number of factors may regulate the rate of movement of receptors back to the cell surface. The rate movement of a receptor back to the cell surface may be detennined by the type of receptor, receptor occupancy, or the endocytic apparatus. The studies in Chapter 3 measure the movement of several different recycling receptors back to the cell surface in a single cell type. The conclusion from these studies is that once internalized, into the same compartment, different receptors move back to the plasma membrane at similar rates independent of occupancy. That is, the rate of movement of receptors back to the cell surface is dependent upon the apparatus or vesicle in which the receptors reside and is independent of the contents of the vesicle. The rate of n10vement of recycling endosomes to the cell surface can be accelerated by treating cells with honnones, which bind to receptors which activate tyrosine kinase or with agents which mimic the inositol phosphate cascade such as phorbol esters or calcium ionophores (Tietze et al. 1980, Buys et al. 1984, 1987, Kaplan et al. 1985, Wiley and Kaplan 1984, Davis and Czech 1986, Ward and Kaplan 1986). The acceleration of vesicle movement is independent of vesicle contents and results in a parallel increase in a number of different receptors on the cell surface. These observations further suggest that these agents affect the vesicle or apparatus itself and not the individual receptors that comprise it. There is an interesting suggestion that while endocytosis can occur throughout the cell surface, recycling vesicles fuse and thus add their membrane to the cell periphery 15 (Bretscher 1984, Hopkins and Trowbridge 1983). Bretscher has used these observations to suggest that the directed fusion of endosomes with the cell swface may provide a means of locomotion for the cell. The Late Endocytic Pathway The exact definition of a late endosome is unclear. These vesicles are the intermediaries between sorting endosomes and lysosomes. Criteria used to identify late endosomes include: vesicles that do not contain Tf or recycling receptors (Mueller and Hubbard 1986), vesicles that do not contain classic lysosomal markers, and vesicles that do contain ligands destined for degradation. Several laboratories have demonstrated the existence of late endosomes. Baenziger and Fiete (1982, 1986) incubated cells in iso-K+ buffers and demonstrated an inhibition in movement of internalized ligand to lysosomes. They extended these studies to demonstrate the existence of receptor-positive (galactose terminal glycoprotein and asialoglycoprotein) and receptor-negative ligand containing endosomes in hepatocytes. Schmid et al. (1988) identified two endosomal populations separated by free-flow electrophoresis and observed different polypeptide compositions for early and late endosomes. The exact origin and trafficking pattern of late endosomes has yet to be identified. It has been shown that some lysosomal enzymes appear to be routed to this compartment prior to their appearance in the lysosome. Binding of lysosomal enzymes synthesized in the Golgi and delivery into a prelysosomal compartment in the endocytic pathway is accomplished by mannose-6-phosphate receptors which are found in the Golgi (Von Figura et aI. 1986, Griffiths et al. 1988, Griffiths 1989). Once the lysosomal enzymes are delivered, these receptors appear to recycle back to the Golgi apparatus for additional rounds of delivery. The mannose-6-phosphate receptors are not found within lysosomes (Von Figura et al. 1986, Pfeffer 1987, Duncan and Kornfeld 1988); therefore, they must be recycled from a late endosomal compartment before the lysosome is reached. Whether 16 the late endosome is a transition state of all vesicles in route to lysosomes or another sorting compartment is unclear (Roederer et al. 1987). The studies presented in Chapter 4 and 5 suggest that it is a sorting compartment. Recent observations have identified endoproteases within the late endosome (Diment and Stahl 1985, Diment et al. 1988, Opresko and Kaarf 1987, Roederer et al. 1987, Doherty et al. 1990). Analysis of isolated late endosomes has demonstrated that they are devoid of most marker enzymes traditionally used to denote lysosomes, such as B-n­acetyl- hexoseaminidase and other acidic glycosidases. However, late endosomes do contain at least one cathepsin, cathepsin D (Diment et al. 1988), an enzyme which was classically thought to reside only in lysosomes. Studies have demonstrated that ligand catabolisn1 n1ay be initiated in this compartment (Diment and Stahl 1985, Diment et al. 1988, Doherty et al. 1990). It is suspected that the late endosome must contain a variety of different proteases since a number of ligands can be digested down to the level of amino acids. Ligand degradation can clearly be initiated in the late endosome; however, on a quantitative basis, only a small fraction of ligand catabolism should occur in this compartment, due to the rapid kinetics of movement from late endosomes to lysosomes. All of these observations lead to a series of questions regarding the function and genesis of this compartment relative to that of the lysosome. If membrane vesicles are fusing with and budding off from late endosomes, and the size of the compartment remains constant, then membrane must also be recycled continually from this compartment. Whether membrane recycling occurs in late endosomes or lysosomes has yet to be determined. Membrane recycling must be occurring along several steps in the endocytic pathway (Griffiths et al. 1989), otherwise, cells may run out of membrane to internalize. In the following chapters an analysis of the movement of receptors and ligands through the endocytic apparatus is presented. A number of questions were examined 17 including rates of movement are receptor specific and what rates are determined by the endocytic apparatus. The rates of movement of different receptors in the same cell type, or the same receptors in a different cell type were examined to distinguish receptor and apparatus specific behaviors. 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Cell BioI. 68:665-680. Stone, O.K., Marnell, M., Yang, Y. and Draper, R.K. 1987. Thermolabile proton­translocating ATPase and pump activities in a clathrin-coated vesicle fraction from an acidification defective chinese hamster cell line. 1. BioI. Chern. 262:9883-9886. Stoorvogel, W., Geuze, H.J., Griffith, J.M. and Strous, G.J. 1988. The pathways of endocytosed transferrin and secretory protein are connected in the trans-Golgi reticulum. J. Cell BioI. 106: 1821-1829. Stossel, T.P. 1974. Phagocytosis. N. Engi. 1. Med. 290:717-723. Tietze, C., Schlesinger, P. and Stahl, P. 1982. Mannose-specific endocytic receptor of alveolar macrophages: Demonstration of two functionally distinct pools of receptors and their role in receptor recycling. 1. Cell BioI. 92:417-424. Tietze, C., Schlessinger, P. and Stahl, P. 1980. Chloroquine and ammonium ion inhibit receptor mediated endocytosis of mannose conjugates by macrophages: Apparent inhibition of receptor recycling. Biochem. Biophys. Res. Commun. 93: 1-8. Ukkonen, P., Lewis, V., Marsh, M., Helenius, A. and Mellman, L 1986. Transport of macrophage Fc receptors and Fc receptor-bound ligands to lysosomes. J. Exp. Med. 163:952-71. VanRenswoude, J.K., Bridges, K.R., Harford, J.B. and Klausner, R.D. 1982. Receptor-mediated endocytosis of transferrin and the uptake of iron in K562 cells: identification of a non-lysosomal acidic compartment Proc. Natl. Acad. Sci. USA. 79: 6186-6190. Von Figura, K., Gieselman, V. and Hasilik:, A. 1986. Lysosomal enzymes and their receptors. Ann. Rev. Biochem. 55:167-193. Wall, D.A., Wilson, G. and Hubbard, A. 1980. The galactose-specific recognition system of mammalian Ii ver: the route of ligand internalization in rat hepatocytes. Cell 21:79-93. Ward, D.M. and Kaplan, 1. 1986. Mitogenic agents induce redistribution of transferrin receptors from internal pools to the cell surface. Biochem.1. 238:721-728. Wiley, H.S. 1985. Receptors and models for the mechanisms of membrane protein turnover and dynamics. Current Topics in Membrane and Transport. Vol 24 pp 369-411. Wiley, H.S. 1988 Anomalous binding of epidermal growth factor to A431 cells is due to the effect of high receptor densities and a saturable endocytic system. J. Cell BioI. 107:801-810. Wiley, H.S. and Cunningham, D.O. 1981. A steady state model for analyzing the cellular binding, internalization and degradation of polypeptide ligand. Cell 25:433-440. Wiley, H.S. and Cunningham, D.D. 1982. The endocytic rate constant. J. BioI. Chern. 257 :4222-4227. 26 Wiley, H.S. and Kaplan, J. 1984. Epidennal growth factor rapidly induces a redistribution of transferrin receptor pools in human fibroblasts. Proc. Natl. Acad. Sci. USA. 81:7456-7460. Willingham, M.C., Maxfield, F.R. and Pastan, I. 1979. Alpha 2 macroglobulin binding to the plasma membrane of cultured fibroblast. Diffuse binding followed by clustering in coated regions. 1. Cell BioI. 82:614-625. Willingham, M.C., Pastan, L, Sahagain, G., Jourdian, G.W. and Neufeld, E.F. 1981. Proc. Natl. Acad. Sci. USA. 78:6967-6971. Xie, X.S., Stone, D.K. and Racker, E. 1983. Determinant of clathrin-coated vesicle acidification. J. BioI. Chern. 258:14834-14838. Yamashiro, D.J., Fluss, S.R. and Maxfield, F.R. 1983. Acidification of endocytic vesicles by an A TP-dependent proton pump. J. Cell BioI. 97:929-934. CHAPTER 2 TIlE RATE OF INTERNALIZA nON OF DIFFERENT RECEPTOR­LIGAND COMPLEXES IN ALVEOLAR MACROPHAGES IS RECEPTOR SPECIFIC 28 1JiocJlem. 1. (1990) m. 369-374 (Pnnted 10 Great Bntaml 369 The rate of internalization of different receptor-ligand complexes in alveolar macrophages is receptor-specific Diane \1cVEY WARD and lerry KAPLAN· Department of Pathology. Cmverslty of Ctah Health Science Center. Salt Lake City. L'T ~413:. CS,A. To probe the m~hamsms of endocytosIS m alveolar macrophages. we exammed the mternalizatlOn rates of three dJlferent receptors. Initial rates of Internalization for mannosylated BSA. dlfernc transfernn and .1;-macroglobuitn-protetnase complexes were all different. Although the absolute rates of mternallzatlon ,aned depending on the ,,;ell preparation. transfernn was mternalized at 10-20'J, and .1;-ma..:roglobuhn-protemase complex at ~40"" otthe rate ofmanosylated­BSA. Incubation 0i cells wah transfernn dId not alfect the rate of mternailzatlon of mannosvlated BSA or .1;-macroglobulin-protemase ..:omplexes. and the rates of mternalizatlon were Independent of receptor ~cupancy. These different mternalizatlon rates could not be .lscnbed to different rates of dlacytosis, Altenng the dlStnbutlon of unoccupied surface receptors by either trypsin treatment of cells at 0 °C or exposure to hyperosmotlc solutions resulted in the absolute internalization rates bemg affected by the expenmental condition. but the hIerarchy m receptor internalization rates was maintained. The fact that a vanety of conditIOns affect receptor mternahzauon rates to the same degree Implies the existence of co-ordinate regulatlon at a Single rate-limiting step. Based on these results. we suggest thal differences m internalization rate reflect the abllllY 01 ligand-receptor complexes to be captured by coated pits. I"'TRODUCTION Receptor-mediated endocytOSIs permItS cells to mternalize macromolecules needed for cellular processes or prOVides a route by which potentially InJunous molecules can be removed from blOloglcal flUids. The receptors \\-hlch mediate these functions are all capable of recycling. Thus. although the ligand may be catabolized. the receplOrs are spared from catabolIsm and may be re-utllized (GoldsteIn e! al .. 1919: .\nderson & Kaplan. 1983) The endocyuc pathway has been characterized usmg both bIochemical and morphological approaches. Most ligand-receptor comple~es are Internalized VIa coated pits. Once Internalized. ligand-receptor comple:o<es are localized In a structure defined as the endosome. The endosome IS an aCidic non-lysosomal ,,;om· partment where uncoupling or dissociation of ligand-receptor complexes occurs (Van Renswoude e! al .. 1982: Harford !?( ell.. 1983: Geuze el ell.. 1983 I. As a consequence of constitutive recycling. the endosome may contain a number of different plasma membrane proteins ;;apable of being recruited 1O the cell surface (Tietze el ai., 1982: Deutsch el al .. 1982: Lamb el al.. 1983: Buys el ai.. 1984) Although the recycling pathway has been descnbed in general outline. a number of questions regarding the movement of molecules through the pathway remain to be answered. We have examined the specific internalization rates oi several ligand-receptor complexes in a single cell type. To date. few studies have examined the rate of receptor-mediated internalization for more than one ligand In a single cell type. Those studies which have done so have usually focused on the Internalization of hormone-receptor complexes. revealtng WIde vanations In internalization rates depending on the particular hormone studied. Measured half-times for the disappearance of surface receptor-ligand complexes range from 2-3 min for epi­dermal growth factor (EGF) In human skin fibroblasts (Wiley & Cunningham. 1982) to 2-3 h for somatostatin in rat pituitary cells (presky & Schonbrunn. /9861. In a number of instances, Internalization of the receptor-hormone complex IS clearly assOCIated with termination of a ligand-mduced signal (Segaloff & Ascoli. 1981). As such. vanations in internalization rate may reveal more about signal transduction than they do about the endocytic machinery To elucidate the factors that may regulate internalization rate we have studied those receptors whose major functIOn IS either to prOVIde reqUired nutrients to cells or to remove tOXII; molecules from biologIcal flUids. Since the function of these receptors IS to accumulate ligand. one can expect that internalization would occur at maXimal rates. Rabbit alveolar macrophages were utilized. as they e,\hlblt a vanety of these 'scavenger' receptors, Our results Indicate that different recrptor-Iigand complexes can be internalized at different rates. As demonstrated by this study and by Ward e( al. ( 19891. mternallzatlon is the only receptor­speCific rate in the recycling process. That IS. once Inside the cell. receptor-ligand complexes or ligands move at rates which are determined by the machinery and not by the receptor or ligand. \1ATERIALS \~D "lETHODS Cells Rabbit alveolar macrophages were obtained by bronchIal lavage (Myrvlk el at.. /961) and the cells were prepared as described previously I Kaplan. 1980) ~.teri.ls .1;-Macroll:lobultn (.1;\-1) was Isolated from either rabbit or human plas-ma. aMyl~I· T was prepared as preVIOusly descnbed IKaplane( al .. 1982). Mannosylated BSA (MAN-BSA) was a gIft from Dr. Y C. Lee. Johns Hopkms University. Baltimore, MD. U,S.A. Maieylated BSA (MAL·BSA) was prepared as descnbed (Butler & Hartley. 197~). Human transfemn (Tf) was obtamed from Calbiochem-Behring (San Diego. CA. U.S.A.), Human Tf was Iron-saturated as descnbed preVIOusly (Ward el al.. 1982). AbbreViations used; Tft Felt. dlfernc transferrm; MAN- BSA. mannosylated bOVine serum albumin. MAL-BSA. maleylated bovlOe serum albumin. aM.T . .x-macroglobulin-trypSIn complexes; HBSS. Hanks balanced salt solullon; MEM. mlOlmal essential medium; EGF, epidermal gruwth factor, • To whom correspondence should be addressed. Vol. 270 370 MAN.8SA. MAL·8SA and Tf(Fe)~ were Iodinated according to the procedure of Markwell ( 1982). Metbotis The binding of aillipnds was measured by the procedure used to assay binding of aMlUI·T (Kaplan. 1980). To distinguish between surface-bound and Internalized ligand. the method of Kaplan and Nielson (19 i 9b) was used for aM.T and \1AN·BSA. However. In the latter case the EDT A buffer emplo)ed was at pH 6.0. A modification of the aCid stnppmg techmque IpH of SA Instead of )8) of Haigler et ai. (1980) was used to distinguish Internalized Tf(Fe)2 from surface-bound TnFel!. Hvperosmouc treatment was performed as descnbed by Heuser & Anderson (1989). Tnchloroaceuc acid precipitatiOn was measured dS de­scnbed preVIOusly (Ward et ai. 1989). Additional procedures Cells were trypsm-treated at O°C for 30 min uSing 0.01 ').) trypSin (Worthington) in Hanks balanced salt solution I HBSS). The reaction was SlOpped by adding a IO-fold excess of soybean trypSIn Inhibitor (Sigma. St. Louis. \10. (J.S:\.). Protein deter­minations were performed as descnbed by Lowry el ai. ( 1951 ). using BSA (fraction V; Sigmal as a standard. RESULTS lateraalization rates of different receptor-ligand complexes Two approaches were used to measure internalization rates of different receptor-ligand complexes. First. the specific internalization rate of receptors (KJ was determined by the method of Opresko & Wiley (1987a). An advantage of this method is that the probability of internahzatJon per occupied receptor can be determined without requlnng changes in In­cubatlOn temperature. We found that Internalization of three different ligands. 1U[·Tf(Fel •. lZ5I-MAN-BSA and aM. wI·T. occurred at different rates (Frg. 1 l. However. the absolute value of the specific internalization rate of a gIven ligand differed with 4oo~------------------------------, 300 ! "C <II !j 200 1 i c ! .: 100 100 200 I/'\Ieoral surflCe ligand (Imol· min) Aa. 1. SpecUk illtenlallzadoa races of aM.lUI·T. ml·MAN·8SA ... lUI-TI'(Fe)1 Cells were Incubated with radiolabelled ligand at 37 'c. At specified tImes cells were placed at 0 0c. washed three times with cold HBSS. and surface-bound ligand was removed as descnbed in the Matenals and methods section. Surface-bound and cell-asSOCiated radio­actiVIty were detemuned and the data were plotted as descnbed by Opreslco &: Wiley (! 987 a) as a function of time. &. IUI-MAN-BSA: .c:... aM.u'I-T; O. IUI.Tf(Fel,. 29 D. McVey Ward and 1. Kaplan different ceil preparallons. For example. the specific mternal­IzatlOn rate of \1AN·BSA vaned by IO-fold In different expenments (see Table 2). Nevertheless. the relative order for internalizatton of the three ligands remained the same: the K. uf Tf(Fel~ was approx. J 5)., that of MAN-BSA. whIle thal oraM.T was 50'), ,)f \1AN-BSA. These results were venfied using an Independent approach. CeJls were mcubated J.t O°C with a\1.ml·T.l~;[-MAN-8SA or lHI_ Tn Fe).! until .;:qUlhbrtum was reached. Cells were washed iree of unbound ligand and mcubated at 37 'c for vanous times. 3fter which surface-bound and internalized ligand was measured. In the presence of divalent cations and at neutral pH. the rates \)/ diSSOCIation of a\1.T or \1AN·8SA and Tf(Fe)~ are e'Hremely slow I Kaplan & 'itelson 1979a: Stahl el ai.. 1980: Lamb et aJ.. 1983). Consequently. the disappearance of surface-bound ligand IS due :-iolely to internalization. These ex:penments confirm prevIous :-i(udles (Stahl el aJ.. 1980. Lamb et ai.. 19831 bv demonstrating thaI the rate of loss of surface-bound ligand exhlbm first-order kmetlcs I Fig. 2). Csing lhlS expenmental approach. we (;onnrm thal 125I-MAN-BSAand aM.12~I-T ex:hlbll 100 90 80 70 ~ 60 50 "0 i. 40 '? '2 :; 30 0 ;:, ~ :; 20 !,/) 10+-----~----~--__ --~ __ --~----~ a Time (min) Fig. 2_ Rates of disappeua.nce of surface-bound·Ugaad Cells were mcubated for 6() mm at 0 "C In the presence of aM. u~f_ T. wPAAN-BSA or ,~.sI_Tf(Fel.. The cells were washed wl!h cold HBSS 10 remove unbound ligand and then placed at 37'C At specIfied tImes ,!liquots were removed and surface-bound and mternalized radlOacttvlIY was determmed as descnbed in Fig. !. The data are plolted ..IS the percentage of surface-bound radIOactivity remamlng as a function orume. &. 1~~I·MAN-BSA. 6. aM.12~I·T: ::::::. 1!~I-Tf(Fe)! Table I. Effect of ligand concentration on the specific internatizaCiotl rate o( 'j~I-\1A"·BSA Cells were Incubated In \1EM at 37 "C with the designated con­centration of wI-MA!'I-BSA. At specified times aliquots were taken and surface· bound and internalized radioactivity was determmed as descnbed In the Matenals and methods secllon The specific tnternaiJzation rate was deterrntned as diSCUSsed by Opresko & Wiley (1987a) and the correlalJon coeffiCient Ir) of the slope IS Included to give an esttmallon of the accuracy of the data. Ligand concentration 04) 139 \.43 1.20 0953 0.977 0.998 1990 Rates of internalization Tallie 1. Eft'edI of Tf'(Fe'2 on the specific internalization rates of li~l_ MAN-BSA and aM.u~I-T The experimental protocol was Identical with that of Table I. except that cells were Incubated with the radlOlabelled ligand 10 the presence or absence of I x IO-~ M-Tf(FeI2 . Ligand Expt. Tf{Fe)z K .. (internaillntegral) U51-MAN-BSA (U~8 O.YK7 0.362 I) YX3 3.030 I).Y75 + ~.Y78 O'l6i aM.'1;I·T I) 151;) n 961 0.15l ilY44 1.050 0.991 1050 1)')74 different internalization rates. The relative differences m rates were similar to the differences in Ke determined above. We did. however. observe the reappearance of 1~51_ Tf on the surface. This result is consistent with the behaviour of Tf. since It IS known that apoTf recycles to the cell surface (Karin & Mintz. 1981). In these experiments, we employed a concentration of ligand equivalent to the K~, or more precisely. the concentration at which steady-state receptor occupancy IS half-ma,'{lmal. In a previous study we demonstrated that aM.T was internalized at a similar rate at either 10 no or 80 0 '1 of surface receptor occupancy (Kaplan & Keogh. [983). As shown in Table I. we obtained . similar results for the MAN·BSA receptor In that the specific internalization rate was similar at both low and high ligand concentrations. Previously we found that incubatIOn of cells with MAN-8SA did not affect the K" of aM.T (Kaplan & Keogh. 1983). Similar results were obtamed With Tr (Table 2). These results suggest that different receptor-ligand complexes are internalized at different rates. and that the rate of internalization of one ligand is unaltered by the Simultaneous internalization of a different receptor-ligand complex. Differences in specific ligand internalization rates are not due to diacytosis It is possible that the differences between the specific internalization rates of disparate receptor-ligand complexes might be due to differences in the rate of intracellular ligand accumulation rather than differences in internalization per se. [f a receptor-ligand complex was internalized and then recycled to the cell surface. our methods of measurement would show a slow . apparent' rate of internalization. The recycling of receptor-ligand complexes. a process referred to as diacytosis or retroendocytosis. has been observed for a wide variety of receptor-ligand complexes (Auslingkas et ai., 1981; Tietze et ai., 1982; Greenspan & St. Clair. 1984; Chang & Kullberg. 1984; Marshall. 1985; McKinley & Wiley. 1988). We measured the amount ofaM.mI·T and mI_ MAN-8SA which was retroendocytosed by giving cells a pulse of ligand. removing surface-bound ligand and measuring the amount of intact ligand returning to the medium. Both cell­associated radioactivity (internalized) and that which was stripped from the cell surface after further incubations were quantified. Acid-precipitable radioactivity in the medium was also measured to assess the degradation of internalized ligand. Approx. 5-15 °0 ligand release was observed following a chase of 2 or 8 min (Fig. 3). It is possible that the stripping procedures affect recycling or diacytosis. In a previous study, however. we compared the rates of recycling of Tf in cells exposed to the stripping procedure versus control cells, and observed no Vol. 270 30 Jil 1 5 Q; 0 5. 10 "5 ~ '0 E 05 ~ 0 C 060 c § 50 . 0.45 "5 ~ 0 0.30 ! 4: CJl CD z 0.15 4: ::i: - 0 C 1 min 2 min 3 min 4 min Fig. 3. Examination of whether internalized IiX.ad is recycled to the eel surface Cells were incubated at 37"C with either aM. 1l5 I-T (a) or ml_ ~A:-.I·BSA (bl for J mm. Cells were then placed at 0 °C and surface­bound radioactivity was removed. The cells were resuspended in Hanks ~EM dnd returned to "C for the speCified times. At each time point the cells were placed at O°C and radioactivity in the media was determmed. Surface-bound (as determmed by the stnp ping technique) and cell-associated radIOactivity were also deter· mmed. The data are normalized to cell protem. C denotes the amount of radioactivity assOCIated with cells after the mitial 3 min mcubation at 37°C; E represents the same sample after stnppmg surface radioactivity. ~. internahzed radioactlvtty at the subsequent incubatIOn i I 4 mlns) at 37 'C: a. the sum of radioactivity In the media as well as that removed from the cell surface difference (Buys et al., 1987) We also measured the rate of return of receptors to the cell surface under conditions In which cells were stripped or not. and agatn saw no effect of the stripptng procedures on recycling rates (Ward et ai., \989). Therefore the results in Fig. 3 indicate that little internalized ligand was recvcled back to the cell surface. and there was no significant difference between the two ligands. The results of these experiments demonstrate that diacytosis cannot explain the observed differences in K •. Topological distributions of receptors are not responsible for differences in internalization rates Differences in internalization rates could be due to differences in the topological distributions of surface receptors. Hopkins ( 1985) demonstrated that in A431 cells Tf receptors were inserted into the membrane at the cell periphery and then had to migrate to coated pits located at some distance away. This observation leads to the hypothesis that differences in internalil;ation rate may be due to differences in the surface distribution of unoccupied 372 400 1 :5 300 ! ~ "S' ~ '! 200 I '0 ~ ) ~ i 0 0 100 200 Fig. 4. :vteasuremenf of K. by surface receptors and those present In the infernal pool Cdls ~ere Incubated wllh saturatlOil aml'unts .11 ¢Ither \1AS-BSA or aMT for 60 mm .,It 37 'C The- -:eHs .. er.:: placed at U T and ~ashed wllh HBSS. The -:e!ls ·.. .. ere then pta..:ed ..II ,; 'C In the presence of either aM.'l;I-T or ";I-\1A,\-BSA. and the specllic internalization rate was determIned JS des..:nbed In the iellend to Fi\!. 1 The speclllC tnternahzallon rate was ..Ilso determtned 7n .::ells Ih~t \l,ere treated Identically <!'l.cept that they had not been <!xposed to non-radIoactive hgand. Internalized ..1M. "'1-T In .:ontroi .:ells (c:) ..Ind tn .:ells exposed to non-radioact!l,<! ligand (.l. Jnd mternaiized IHI-MA:-.I-BSA 10 control .:ells (L . i Jnd 10 .:e1i5 e'l.posed to the non­radioacllve ligand IA). was measured. receptors. Some specIes of receptors may be located at or near coated pits. whereas others mIght be randomly dtstrlbuted 3.nd therefore have a greater distance to mIgrate to coated PItS. To test thIS Idea. three different approaches ""ere taken to alter the surface distnbullon of receptors. [nternalizatlOn of unoccupied receptors lS slower than mternallzatlon of occupied receptors. and. after exposure of cells to ligand there IS a marked reductIOn m the number of surface of receptors. most receptors beIng present In the mternal pool (Ward /!t al.. 1989). The rate of dISSOCIation of ~AN-BSA or aM.T is so slow that ligand does not diSSOCIate from surface receptors even at 37 ~c. Thus. in cells exposed to non-radioacllve ligand. uptake of radioactive ligand would only be mediated by receptors that had previously been WlthlO the cell. Exposure of cells to non-radlOacllve ligand results in a 90"'.) reducllon \0 surface receptor number. This was determtned by removtng surface-bound non-radioactive ligand followed by measurement of the binding of radioactive ligand.' Most remaming receptors were occupied by ligand. as determmed by the addition of radioactive ligand without stnpplOg. Thus less than 1.0 Q [) of the original surface receptors were unoccupied. Because most are inside the cell at the start of the e:\penment (addition of radioactive ligand). there should not be any inherent difference in surface receptor distribution. The rate of internalization. mediated by receptors that had been withtn the cell. was identical with that carried out by receptors present on the surface (Fig. 4). A Similar result was obtained using a different protocol. Cells were treated with 0.01 "" trypslO at 0 'c. This procedure destroys mannose-terminal glycoprotein receptors but spares those receptors localized withm the endocyuc apparatus (Stahl et aJ.. 1980). aM. P receptors are not sensitive to trypsm treatment and therefore serve as a control to Insure that trypslO treatment does not alter endocyttc activity per H'. Followmg trypsin treatment. cells were placed at 37 'c and the K,. was measured to demonstrate 31 D. \1cVe\ Ward .Ind J KJplan 300;---------------________________ ~ I 200 ";l ;; '1 g ~ ¥ 'no ~ )~--------------------------~----~ 'J , 00 200 :rtegrai .yrface ligand \ fmol· min I Fig. ~. Effect of tr~psin on mrernationalization rate C .:Ils were Incuhat.:d J[ t) ?C for 10 mm In the presence or Jh~.:nc.: elf 001 ' .. nl, pSln and the ,ells were washed With H ass .:ontalntnll t) 1, 'io~bean trypsin inhibitor. Cells were shIfted to 37 'C 10 Ih~ presenl:':: of JM. "'~I-T or W(-'vfA:-'-BSA. ,\t specified times . ..:~:Is w.:re plal:ed at O°C Jnd washed wlIh HBSS. and tnt.::rnahzed .lnd surra.:e-bound radioacli\ltv \l,as determmed. InternalIzed hiland was cakulated as descnbed In the legend to Fig. I. Internahzli'tton riots of ,".II-'v1AS·BSA iO control I..:':;.) and trypsIn-treated IA) ~t:'ls. Jnd or J~ . .,.I[_T in -:ontrol (e) Jnd trypsin-treated cells '.) are shown. that thiS manoeu, re did not alter endocvtlc activItv i Fill. 5). Even though trypsin tr":Oitment destroyed 90", 01 :iu~face- receptors i results not ,hown I. the Ke of 'HI-MAN-BSA was Similar 10 trYPsin-treated J,nd control cells. A potential ..:a ... eat to the above experiments is that the rate at which unoccupied receptors regain their ongmal surface diS­tribution might be taster than the rate of ligand bmdinll (Wilev. 1985) Thus~ pnor to the btndmg of lig;nd to receptor. the receptors may h..!ve .llready regained their differenual distn­butlOn. To test thiS pOSSibIlity we employed a condition ""htch randomizes surface receptors. [ncuhatlon of cells In hyperosmotic medium results tn .In mhlbitlon 01' receptor-medIated mternalizatlon, Daukas & Zilm1ond. 1985. Oka & Weil!el. 1988) Correlated wah thiS IOhlhitlo~ is the disappearance of c~ated pitS from the ..:ell o;urface Jnd .l random dlstnbutlon of previously clustered surface receptors I Heuser & Anderson. 1989). We took advantage of thiS observation to effect differences m internalization r.ltes. Ceils were Incubated at O°C with Il,,[_ \1A~-BSA or J.'v1. lll [_T to occupy surface receptors either tn Iso-osmotlc medium or in medium made h~perosmotlc by the addition of sucrose. The cells were then washed and Incubated at 37°C in either h;.per- or Iso-osmotic medium and the rate of internalizatIOn was meOisured. [n hyperosmotlc medium there was a marked mhlbltlon of the mternalizatlon of both ligands (Fig. 6£1 versus Fig. 6b). Electron micrographs of wntrol and hyperosmotlc-treated cells were analysed for coated pitS (results not shown J. Few observable coated pitS were seen In cells treated WIth hyperosmotlc buffers (approx. 5-15", 01 control). When cells preVIOusly tncubated 10 hyper-osmotic medium were incubated in Iso-osmotiC medium. Internalization recovered dur­ing the time course measured. although not to normal levels. The speCIfic tnternalizatlon rate ol'~';[-\1AN-BSA. however. was stili faster than that of a.'v1. 1'''[-T. These results suggest that the difference in Internalization rates IS not due to differences in surface receptor dlsmbution. 1990 Rates of interna.tization l "S1 iii ~ ~ 5 .0 ~ S Vl l00~------------__________________ ~ 90 80 70 50 50 40 30 20 10 100 90 80 70 60 50 40 30 20 [al 0 b\ 10+--.----~--~~--_r--------~~----~ o 4 5 Time {m[nl Fig. 6. Effect of byperosmotk treatment Oft me internalization rates of aM. m(·T aDd 'l~I·!VIAN-DG" Cells were incubated In either Iso·osmotic or hyperosmotlc bulfers (045 ... -sucrose In Hanks ~EM I at 37 'c for 10 min. The cells were then placed .it 0 'c. Incubated wnh ligand. washed extenslve!:- and shlrted to 37"C. and at the indicated umes surface as welt as internalized radIOactivity was measured as descnbed In the legend to Fig. ~. la) Cells maintained In Iso-osmotic media. Ihl cells enher mamtalned In hyperosmollc bulfers throughoul the expenmenl (.) or Incubated In hyperosomotlc buffers and then placed In Iso-omotlc buffers (Al. IU!·MAN·BSA. 6. aM.ll~I·T) Dlsa;SSION We considered a number of different explanations for receptor­spec1fic internalization rates. Differences 10 IOternalization could result from the eltistence of ligand-speCIfic coated pits. each of which is internalized with a charactenstic rate constant. Even though recent studies suggest this possibility (Gonnan & Poretz. 1987: Goldberg et at.. 1987). two lines of evidence make this unlikely. First. morphological studies have demonstrated that more than one type of receptor-ligand complex. can enter the same coated pit (Carpentier et al.. 1982). Second. we have demon­strated using the horseradish perox.ldase I diammobenzidine technique that there are no ligand-specIfic endocyuc veSIcles in two different cell types using five different ligands (AJioka & Kaplan. 1987, Ward et ai.. (989). These observations do not role out the POSSIbIlity that ligand-specific coated pus fuse Vol. 270 32 rapIdly after internalizatIOn. There are studies whIch demonstrate fUSion In l'ICO among earh endosomes I Salzman & \1axheld. 1988. Ward et al., 1990). We think that the number of receptors IS $uch that It IS unlike!;. that there are a large number of receptor-speclfic coated pitS. DIfferences In mternaltzauon rates could result from Inherent differences tn the surface receptor distnbutlOn For eumple, certam specIes uf receptors may be locailzed m coated pits. whereas others may be randomlv distnbuted. and differences In mternallzatlon rate may reflect the tIme reqUIred for the random!\! dlstnbuted receptor to mIgrate to coated pits. -\ number 0f experlmemal approaches "rgue agatnst thIS po-:;slbllltv. There were no differences In the SpeClhC InternalizatIOn rates exhIbited r,v receptors present on the surface 3.nd those that had been '>'Ilhm the \!ndocytlc .Jpparatus. AdditIOnally. the difference In relatne internalIzation rates perSisted when cells were placed under conditions In which surface receptor Jlsmbutlon was randomIzed. A third pOSSIbIlity IS that differences m internalizatIon rate mav reflect differences In diffusion rates between random Iv dlstnbuted receptors. Although there are no direct data that would rule out thIS possibIlity. several conSIderations make It unlikely. There IS little eVIdence that diffUSIon times are rate­limiting for endocytOSIs (Goldstein & Weigel. ! 983. Wdey. 1985). For a given membrane molecule. large structural deletions in either the cytoplasmic or the external domain have little etfect on the diffUSion rate (Lehnnann el al .. 1985; Prywes et al .. 1986): suggestmg that lateral diffusion rates of receptors are not related to or affected by the size of the membrane protem or that of the ligand. The fourth pOSSIbility. and the one that we favour. is that differences In Internalization rates reflect differences In the . capture' rate ot' receptors by the coated pit. For example. membrane protems may be freely mobile, but If they cannot duster m coated pitS they are unable to be internalized / Anderson er al .. 1981 ) The~e protems could either be excluded from coated PItS. by bemg Immobilized at a distal site. or are Simply unable to be captured by coated PItS. \1utants m the cytoplasmIC domain of the low-denslty·lipoprotetn receptor eXIst in which the only observable etfect IS a lack of localization m coated pitS \ Lehnnann et al. (985) Similarly, some mutants 10 the cyto­plasmiC domam of the EGF receptor also exhIbit an inability to both cluster in coated pItS and be mternalized t Prywes t!t al .. 1986) Studies demonstrate the eXistence of freely dlffusable membrane protems which .ire not internalized dshihara t!l at. 1988). These observations suggest that internalization does not reflect a default pathway. but rather is the result of an interaction between membrane receptors and elements pre3ent m the coated pit. Severa1 groups ha've Identtried protems aSSOCiated with coated PIts {'adaptms': c1athrtn-associated proteIns) whIch are thought to medIate the bmdlng of receplors {Cnanue III al .. 1981. Pearse & Bretscher. 1981. Pearse. ! 985. Prasad & Llppoldt. 1988. Virshup & Bennett 1988: Ahle & Cngewlckell. 1989). However. to date there IS little mfonnatlOn on the affinity of receptors for these adapttns or on the stoichiometry of adapuns and receptors. PreVIOusly we demonstrated that the internalization of large particles such as zymosan or IgG-coated red blood cells by alveolar macrophages did not affect the Internalization rate of a number ot" different recycling receptors. These observations are SImilar to those of Ciechano\!er el i.J./. (1983). who demonstrated that in cultured hepatoma cells internalization of aSlaloglyco­proteins and Tf were mdependent of each other and of receptor occupancy. An mability to saturate imernalizatlon might seem to argue against a role for adapttns. Alternatively_ the concentration of receptors may be such that under most conditions bindmg of receptors to adaptins IS not rate-limiting. Analysis of the literature 374 reveals two reports in which the specific internalization rate constant decreases with receptor occupancy. The specific internalization rate of vitelligenin by Xenopus oocytes and of EGF by cultured A43l cells decreases with receptor occupancy (Opresko & Wiley, 1987b; Wiley, 1988). Even at receptor occu­pancies at which the rate of EGF receptor internalization becomes markedly decreased there is no effect on the inter­nalization of transferrin. It was suggested that saturation of receptors within the endocytic apparatus only occurs at extremely high receptor densities. For example, there is no evidence of saturable internalization of EGF receptors in fibroblasts, a cell type in which the density of EGF receptors is three orders of magnitude lower than in A431 cells. The second conclusion is that different receptors may interact with different components in coated pits, i.e. different adapters, which may vary to con­centration. Our data also indicate that there are elements extrinsic to receptor molecules which are Important m specifying internalization rates. We found wide variations to the absolute rate of ligand internalization with different cell preparations. Even though the absolute rate of ligand accumulation varied. the hierarchical distinctions in mternalization were maintained. The cells used in this study were obtamed from rabbits that had been injected with Freund's complete adjuvant. This agent produces massive pulmonary granulomas and results 10 a IO-Ioo-fold increase in cell number due to the recruitment of blood monocytes into the lung. While in the lung these cells undergo a number of biochemical change, which distinguish them from blood monocytes or other macrophages. Among such changes are increases in aerobic metabolism, lysosomal enzyme levels and bacteriocidal activity. We suspect that the difference in absolute rate may reflect cells at different developmental stages. Differences in absolute rates of internalization have been observed for the low-density-lipoprotein receptor introduced by genetic manipulation mto human or hamster cells (Beisiegel et ai., 1981). InternalizatIOn and clustering of the low-density­lipoprotein receptor differs between fibroblasts and epithelial cells (Anderson et ai., 1981). The observation that the same receptor molecule (gene) behaves differently in different cell types clearly indicates that there is some feature extrinsic to the receptor which regulates internalization. The fact that one can introduce the same receptor into two different cells or examme two different receptors in the same cell offers an opportunity to define those factors. We than': Stephante Hamilton and Mona McArdle Cor technical assistance and Sandra R. DaVIS Kaplan for electron microscopy. We also thank Dr. H. S. Wiley and Dr. M. Rechsteiner for their useful conversations and advice. This work was supported by a grant from the NatIOnal Institutes of Health (HL26922 and DK3(435). REFERENCES Ahle, S. & Ungewickell. E. (1989 J. BioI. Chern. 264. 20089-20093 Ajioka. R. S. &. Kaplan. J. (1987) J. Cell BioI. 104, 77-9\ Anderson. R. G. W. & Kaplan, 1. (1983) Mod. Cell BioI. l. 1-5\ Anderson, R. G. W. Brown, M. S. & Goldstem.1. L. (1981) J, Cell BioI. 88,411-452 Auslingkas. T. H .. Van der Westhuyzen, D. R .. Bieran. E. L.. Gevers, W. & Goetzee, G. A. (1981) Biochlrn. Biophys. Acta 664, 225-265 Beisiegel. U .. Kita. T.. Anderson, R. G. W., Schneider, W.1., Brown. M. S. &. Goldstein. J. L. (1981) J. BioI. Chern, Z!6,4071--4078 Butler. P. J. G. & Hartley, B. S. (1972) Methods Enzyrno!. 25. 191-199 Buys. A. A. & Kaplan. 1 (1987) 1. Cell Phys. 131. 442-449 Buys. S. S .• Keogh. E. A. & Kaplan, 1 (\ 984) Cell 38. 569-576 Received 21anuary 1990/9 May 1990; accepted 11 May 1990 33 D. McVey Ward and J. Kaplan Buys. S. S. Gren. L. H. & Kaplan. J. (1987) 1 BIOI. Chern. 262. 12970-12976 Carpentier. 1. L Gorden. P. Anderson. R. G. W, Goldstern. J L. Brown. M. S. Cohn, S. & Orci, L. ([982) J. Cell Biol.~. 7J-77 Chang. T. & Kullberg, D. W. i 1984) Blochirn. BlOphys. Acta 805, ~68-276 Ciechanover. A .. Schwartz. A. L.. Dautrv- Varsat. A. & Lodlsh, H. F. ( 1983) J. BioI. Chern. 258. 968 1-9689 . Daukas. G. & Zigrnond, S. H. (1985)1. Cell BIOI. 101. 1673-1679 Deutsch. R. J,. Rosen. O. M. & Rubin. C. S. (198~)1 BIOi. Chem. 257. 5350-5358 Geuze,1. 1.. Slot. J. W .. Strous. G. J. A. M .. Lodish, H. F. & Schwartz, A. L. 1(983) Cell 22. 277-~87 Goldberg. R. l.. Smith. R. M. & Jarett, 1. (1987) 1. Cell. Phys. 133. 203·212 . GoldsteIn, B. & Weigel. R. W. (1983) Biophys. 1. 43. 121-125 Goldstein. 1. L.. Anderson. R. G. W & Brown, M. S. (1979) !'Iature (London) 279.629-685 Gorman. R. M. & Poretz, R. D. (1987) 1. Cell. Phvs. 131. 158-164 Greenspan, P & S1. Clair, R. W. i198411 BioI. Chern. 259,1703-1713 Haigler. 1. T.. Max.field, F. R. & Pastan, L (1980) 1. BioI. Chern. 255. 1239-1241 Harford. L Bridges, K R .. Ashwell. G. & Klausner. R. D. (1983) 1 BioI. Chern. 2.58. 319\-3197 Heuser. 1 & Anderson, R. G. W. (1989) J. Cell BioI. 108.389--400 Hopkins, C. R. (1985) Cell 40, 199-208 Ishihara. A. Holifield. B. & Jacobson. K. (! 988) J Cell BioI. 106. 329-343 Kaplan. J (1980) Cell 19. 197-205 Kaplan, 1. & Keogh. E. A. i 1983) Ann. N.Y. Acad. Sci. 421. 442-456 Kaplan, J. & Nielsen, M. N. (1979a) 1. BioI. Chern. 2.54, "'329-7335 Kaplan J. & Nielsen. M. N. (1979b) 1. BioI. Chern. 2.54. 7335-7340 Kaplan, J .. Ray. F.D. & Keogh. E.A. (1982) 1. BioI. Chern. 2.56 •.. 7705-7707 Kann. M. & Mintz, B. (1981) 1. BioI. Chern. 2S6, 3245-3251 Lamb. 1. E .. Ray. R., Ward. l H .. Kushner, 1. P & Kaplan, J. (1983) J. BioI. Chern. 258, 8751-8758 Lehrmann. M. A .. Goldstem, 1. c.. Brown, M. S., Russell. D. W. & SchneIder. W 1.11985) Cell 41. 735-743 Lowry. 0. H .. R05ebrough. N. J .. Farr. A. L. & Randall. R.1. (1951 f J BioI. Chern. 193. 265-275 Markwell. M. (1982) Anal. Biochern. 125.427432 Marshall. S. (1985) 1. BIOI. Chern. 260,13524--13531 McKinley. D. "I. & Wiley. H. S. (1988) J. Cell. Physio!. 136. 389-397 Myrvik, Q. M .. Leak, E. S. & Farris. B (1961) J. Irnrnunol. 86,133-136 Oka. 1. A. & Weigel. P H. i 19881l Cdl. Biochern. 36. 169-183 Opreski. L. & Wiley. H. S. (l987u) 1. BioI. Chern. 262,4109--4115 Opresko. L. & Wiley, H. S. (1987h) 1. BioI. Chern. 262.4116--4123- Pear5e. B. M. F. i 1985) EMBO J. 4, 2457-2460 Pearse. B. M. F. & Bretscher, M. S. (1981) Annu. Rev. Biochern. SO, 85-101 Prasad, K. & Lippoldt. R E. (1988) Biochemistry 27.6098-6104 Presky. D. H. & Schonbrunn, A. ! 1986) 1. Cell BioI. 102.878-888 Prywes. R .. Livenh. E. Ullrich. A & Schlessiger. 1. (1986) EM BO 1 5. 2179-2190 Salzman, 1. N. & F. R. Max.field. (1988) 1. Cell BioI. 106. 1083-1091 Segaloff. D. L. & Ascoli. M. (1981) J. BioI. Chern. Z!6. 11420-11423 Stahl. P., Schlessinger. PH .. Sigardson. E.. Rodman. 1. 1. & Lee. Y C. (1980) Ceil 19. 207 -215 Tietze. c., Schlessmger. P & Stahl. P (1982) J. Cell BioI. 92. 419--424 Unanue. E. R., Ungewlckell. E. & Branton. D. (1981) Cell 26. 439--W6 Van Renswoude. L Brtdges. K. R .. Harford. J B. & Klausner, R. D (1982) Proc. NatL Acad. Sci. U.SA 79. 6186-<:l190 Virshup, D. M. & Bennett. V. (1988) J. Cell BioI. 106.39-50 Ward. 1. H .. Kushner, J. P & Kaplan, 1. (1982) 1. BioI. Chern. 257. 10317-10323 Ward, D. M., Ajioka, R. S. & Kaplan, 1. (1989) J. BioI. Chern. 264. 8164-8170 Ward, D. M., Hackenyos. D. & Kaplan, J. (1990) 1. Cell BioI. ItO. 1013-1021 Wiley, H. S. (1985) Curro Top. Mernbr. Transp. 24, 369-411 Wiley. H. S. (1988) 1. Cell BioI. 107.801-810 Wiley, H. S. & Cunningham, D. D. (1982) J. BioI. Chern. 257.4222-4227 1990 CHAPTER 3 COHORT MOVEME~l OF DIFFERENT LIGANDS AND RECEPTORS IN TIIE INTRACELLULAR ENDOCYTIC PA TIlWA Y OF ALVEOLAR MACROPHAGES 35 T'H& ,Jot:.".u. 01/ ..... OGICAL CHEMIST1!Y e L_ by "I"ht .-.- ~l.tv (or BlOcn~mISt" and \Iolecu!ar BloiollY Inc Cohort Movement of Different Ligands and Receptors in the Intracellular Endocytic Pathway of Alveolar Macrophages* I Received tor puhlicatlOn, ,',fa" 10. 19~1:! I Diane McVey Ward. Richard Ajioka. and Jerry Kaplanl The rate of movement of different receptors and ligands through the intracellular endocytic apparatus was studied in alveolar macrophages. Cells were ex­posed to iodinated a-macroglobulin-protease com­plexes. mannose terminal glycoproteins. diferric transferrin. and maleylated proteins. By use of the diaminobenzidine density shift procedure, we demon­strated that these ligands were internalized into the same endocytic vesicle. We then compared the rates of transfer to the lysosome or recycling to the cell surface of different ligands/receptors contained in the same endosome. We found that although the rate constant for degradation was ligand specific. the lag time prior to the initiation of degradation was the same for aU three ligands. We also found that molecules taken up nonspecificaily by fluid-phase pinocytosis had the same lag time prior to degradation as ligands internalized via receptor-mediated endocytosis. These data suggest that different molecules within the same endocytic compartment are transferred to the lysosome (or deg­radative compartment) at the same rate. We measured the rate of return of receptors to the cell surface by either inactivating surface receptors by protease treat­ment at 0 <Ie, or by incubating cells with saturating amounts of nonradioactive ligand at 37 <Ie. We then measured the rate of appearance of "new" receptors on the cell surface. Using these approaches. we found that three different receptors were transferred from inter­nal pools to the cell surface at the same rate. The rate of transfer was independent of whether receptors were initially occupied or unoccupied. Our observations in­dicate that receptor/ligands. once inside alveolar mac­rophages. are transported by vesicles which transfer their contents as a cohort from one compartment to another. The rate of movement of these receptors is determined by the movement of vesicles and is inde­pendent of their content. The recycling pathway involves a number of discrete events such as internalization of receptors. dissociation of internal­ized receptor-ligand complexes, and the return of receptors back to the cell surface. While recycling is a continuous process. the rate of movement of molecules through different parts of the pathway can be modulated by either pharmaco­logical agents or alterations in environmental conditions (Buys et ai .. 19841. For example. the rate of movement of • This work was supported Ln part by Grants HL2692:205 and DK35034 from the ~ational Institutes of Health. The costs of pub­lication of this article were defrayed In part by the payment or page charges. This article must therefore be hereby marked "adl;erttse­men£" in accordance With 18 C,5,C. SectIOn [734 solely to mdicate this fact. + To whom correspondence should be addressed. receptor!> trom internal compartments to the cell surface lS more sensItive to changes tn temperature and A TP content than is mternalization ! Weigel and Oka. 1981; Clarke and \Veigel. 1982). Dissoclatlono!' mternalized ligand-receptor complexes or recycling back to the surface is relatively more 5ensitive to changes in the pH of the intracellular compart­ment than is internalizatIOn (Kaplan and Keogh. 1981: Tietze et at.. 1980: Gonzalez':'';oriega et ai.. 1980). Internalization appears more sensitive to hypo-osmolarity than is recycling I ~ovak et ai., 1988). These manipulations demonstrate that intracellular ligand movement is compartmentalized and have allowed experimental alterations of one 11mb of the pathway relative to others. One way to analyze the endocytic pathway is to determine whether all the compartments in the pathway behave the same. and if not. what features of either the receptor or the endocytic apparatus contribute to the differences. In a study which will be reported elsewhere. we demonstrated that the rate of internalization was receptor specific.' The questions we posed in the present study were, is the rate of movement of molecules through intracellular compartments receptor speCific? Do different ligands or different receptors exhibit characteristic rate constants for eIther transfer to the lyso­some, or movement back to the cell surface? Different rates of intracellular movement would be consistent with receptors entering and exiting a central compartment in which move­ment through the compartment would allow for different rate constants. Alternatively, Similar rates of movement through the endocytic pathway would be more consistent with bulk transfer of components from one compartment to another. The latter behavior could best be explained by vesicular transfer in which all components were transferred as a cohort. Our results uSing two separate approaches I measurement of the time it takes for internalized ligands to reach the lysosome. and the time it takes for receptors to return back to the cell surface) indicate simiiar rates of movement for different receptors or ligands. These results suggest cohort behavior for molecules once internalized and are consistent with the VIew that the endocytic apparatus behaves as a series of vesicles, the contents of which are not mixed. EXPERIMENTAL PROCEDURES Cells- Rabbit .1lveolar macrophal\es were obtained bv bronchial lavage L \tyrvlk et al.. 19611 and the cells prepared as deSCribed prevlOuslv L Kaplan. t980L .Watertals-<l"\tacroglobulin was isolated from eLther rabbit or numan plasma and Ct-\t· "'1-T' was prepared as preVIOusly deSCribed I Kaplan et al.. [9821. \1annosvlated-bovine serum albumin (\fA~- , D. \1. \Vard and ·1. Kaplan. manuscript in preparation, ! The abbrevLations used are: Ct- \-f. T. Ly-macroglobuhn·trypsin complexes: \fAN' oSSA. mannosvlated-bovlne serum albumin; MAL­BSA. malevlated-bovlne serum albumin; MTG, mannose terminal glycoprotel~; DAB, diamlnooenZidine 8164 36 Intracellular Trafficking in A.lveolar }v!acrophages 816,) BSAI was a ~Jt't trom Dr. Y C. Lee. Department of Biology. ·Johns HopkIns t·nlversitv. \laleyiated·bovine "erum albumin 1 \IAL·BSAI was prepared as descnbed by Butler and Hartlev 1197:21. Human diferric transferrin wa,; obtained from Calbiochem. Human diierric transfernn was Iron saturated as described previously I Ward et al .• 191".21, \IA:'-i·BSA and diferric transierrimFel: were iodinated ae· cordIng to the procedure of Markwell i 198:21 J/ethlJd.,-The hinding of all ligands was measured as described (or the bIndIng of ,,·\1· :"'I·T I Kaplan. 198();. The concentration of ligand emplu\'ed wa".1 x lIr' \{ for d·\l·"l·T 1 10-' \{ for \lTG. 1 x 10-- \{ I'or difemc transfernn. and L x to-' \{ lor \IAL·BSA. The "pecltic rate of internalizatton IS mdependent 01 the receptor <)C("llrllln,cv for the;;e ligands: The method or' Kaplan and :'-iielson was used to distingUIsh between surtace bound and Internalized ft· \1· T, A simIlar protocol was used for \L-\)i·BSA USIng a buffer of pH /).0, The acid·,uipping technique ot HaIgler ai. \ 1980 I was used to distinguIsh Internalized ;c'r·difernc transternn from wrface-bound ligand wllh the modification that the pH "f the buller was ;;A. To measure the generation of ligand del!radatlOn products. supernatants from binding assavs were added to an equal volume of lOCe tnchio· roaCE'tlc aCId. incubated at 0 'C for :10 min. and centrifuged at ,.,00 x /.t for 10 min. Radioactiv1lv 10 the ;<upernatant was then measured. .Xl! experiments were done a minimum or' three urnes. and represent· ative experIments are demonstrated. Addwonai Prr)Cedures- Protein determinattOns were performed as descrIbed bv Lowrv et al. 119:i 11 using novine serum albumlO t traction \' ~i~ma I as 5tandard. Ceils were trvpslOlzed WIt h 0.0 1 r~ tr\-psin I Wort hington. :\lalvern. PA) in Hanks' balanced salt solutIOn. Celis were mcubated with the tf\-psin solution for ,30 min at 0 ·C. and the reaction was termmated by adding an equal volume of soybean trvpsm inhibitor 1 Sigma!. The acti\'ities ot alkaline phosphodiester· ase and .j·.V·acetvlhexoseamlOidase were measured as described bv Edelson and Erb~ Il978) and Kaplan 1.19781. respectively, \lethods for employing the DAB/density shift procedure. including the prep­aration of Percoll gradients. are described by Ajioka and Kaplan i 198;'L Percoll density was measured at :.::.: 'c using a Bausch and Lomb refractometer. RESLLTS Localization of Different Ligands in the Same Endosome­Rabbit alveolar macrophages contain several receptors capa­hIe of mediating ligand accumulation. Included among these are ~TG receptors, diferric transferrin receptors, a-M-pro­tease receptors, and the "scavenger" receptor which mediates uptake of maleylated-bovine serum albumin and altered low density lipoproteins (Brown et al.. 1980). The number of receptors/alveolar macrophage is approximately 1 X 105 with the exception of the diferric transferrin receptors which are approximately 10-fold lower. The amount of ligand required for half-maximal occupancy (Kd ) is similar for these receptors varying between 0.5-1.0 X 10-; M. Although these receptors mediate the uptake of different ligands they all are capable of recycling. Morphological studies demonstrate the presence of more than one receptor-ligand complex in a coated pit. recent studies indicate there may be receptor-specific endosomes (Gorman and Poretz. 1987; Goldberg et aI.. 1987). To define the localization of different ligands in alveolar macrophages. we employed Percoll gradient centrifugation of cellular ho­mogenates. After 3 min of exposure to a-M· 125I_T the distri­bution of radioactivity was coincident with the plasma mem­brane ectoenzyme alkaline phosphodiesterase (Fig. 1A). When cells were incubated at 37 'C for longer periods of time, radioactivity was found coincident with the lysosomal marker, i3-N·acetylhexoseaminidase (Fig. IBl. Identical results were observed for ,2"I-MAN -BSA (data not shown). These results. however, do not prove that both ligands are in the same compartment. To directly address this point we used the horseradish peroxidase/density shift technique established by Courtoy et ai. (1984) and modified by Ajioka and Kaplan ( 1987). Horseradish peroxidase has commonly been employed as a fluid-phaser marker in a number of cell types. However, (A) 3 I 14 -10 20 .! 110 2 ... 8 g 106 z"7' • .::.. i 10 ... 1.02 4 52 '0 . • ::I . ., &.2 is .!. :I 2 ,,: e I- »" \J · ~ \J l- I- - I- ~ e 1ft ! u CIt ~ C .... u ".: :•:I <wII c 1.14 =: 50~ </I I ~IO z w c u "LlO ~ I- 0 Z <wII 8 40! I 2 c 1.06 6 30~ 0 )I( 1.02 4 20 ~ W c~ 2 10 ~ ~ c 5 10 15 20 FRACTION NUMlER FIG. l. Distribution of a-M. 12~I_T. alkaline phosphodiester­ase. and ,8.N·acetylhexoseaminidase on Percoll gradients. Cells were mcubated with "·\I·,,sl-T at ;j7'C for:1 mm IAI. The cells were then placed at 0 'C and surface·bound ligand removed. One aliquot was placed back at :37 'C for an additional :30 min in the absence of ligand i B I. Cells were homogenized in 0.:25 M sucrose in lO mM Tris·HC!. pH 7.:2 .. ') m\{ EDTA and centrifuged at 800 x g tor 10 min. The supernatant was applied to :.:ire Pereoll and centrifuged at 59.000 x g for 27 ffilO. Gradients were fractionated and assayed for radioactivity. denSity. and enzvme activities. C . .v represent~ tY·~l. 125 1_ T: e, .v alkaline phosphodiesterase actIvity; 6 . .v hexoseamlOi· dase activity .•. .v densitv. in macrophages. it is also internalized by the MTG receptor and at low concentrations can be used as a ligand to study receptor behavior (Kaplan. 1980). DAB diffuses into mem­brane vesicles where intraluminal horseradish peroxidase cat­alyzes the formation of a dense polymer of DAB. increasmg the buoyant density of the vesicle and shifting it to denser regions on either Percoll or sucrose gradients. Since the shift in density is restricted to those vesicles that contain horse­radish peroxidase. a concomitant change in density of a marker indicates its localization in the same compartment. To determine if 125[·MAN-BSA and a-M· i25[_T were in the same endosome, cells were incubated with a· M· 1251. T and horseradish peroxidase for 3 min at .37 'C and then placed at 0°(, Horseradish peroxidase, at this concentration (2.5 X 10-8 M). is taken up predominantly by receptor-mediated endocytosis. Surface-bound ligand was then removed, cells homogenized. and the homogenate applied to a Percoll gra­dient, Selected fractions of the gradient were removed and incubated in vitro with DAB/H20~ before application to a second Percoll gradient. As a control, aliquots of cells incu­bated separately with either horseradish peroxidase or a-M· 1251_ T were mixed and homogenized. Only the sample obtained from cells incubated simultaneously with both ligands exhib­itee) a density shift (Fig. 2, A and B). As demonstrated 37 8166 Intracellular Trafficking in Alceolar iHacrophages (A) z .. ----------------~ 10 15 20 (C) ~'.I. ~ u ~I 10 u i ""06:: iii ·,02 ~ ~ 1'"----------_1 I. ft ~ 2. 110 :, ~4 lOll I :: :-2 lot i !l! ~ ~ 10 15 20 (B) 2 \ !. .\ 1"114 ! 2 /1 7 ~ I 10 ~ ... e \~~ .. ClIO .. -106 = 2 I . Vi • o /= I Z 2 ",I 02 ~ 0- ;) ... .,....,'" .,,,-,,' U 10 20 (0) -. 2 ..,.-----------.., '0 15 20 I 14 .! 110 .'".. e ClIO 106 = izii 102 ~ FRACTION NUMIER FIG. 2. Distribution on Percoll gradients of a·M.1uI·T and 12111-MAN·BSA after a density shift. Cells were incubated with either lI5I-MAN-BSA_ (t_:'vl. 123I_T. or horseradish peroxidase. A second group of cells were incubated with lI5I-MAN-BSA + horseradish peroxidase. or a_M· 125I_T + horseradish peroxidase at 37'C for 3 min. The cells were then placed at 0 'C and surface-bound ligand removed. Cells were homogenized and prepared as described in Fig. 2. Control samples. incubated with only radioactive ligand or only horseradish peroxidase. were combined after homogenization. The supernatant was applied to 27% Percoll and centrifuged at 59,000 x g for 27 min. Gradients were fractionated and assayed for radioactivity (data not shown!. Radioactive peaks were pooled I fractions 9-11) and samples were incubated with HzOz and diaminobenzidine as described under "Experimental Procedures." The samples were then reapplied to a 27% Percoll gradient. centrifuged at 59.000 x g for 27 min. fractionated. and radioactivity determined. A and C represent control I mixing) samples. Band D represent cells Incubated with both horseradish peroxidase and a-M .ll51_T or 125I-MAN-BSA. respectively. O. a_M. l251_T; e, l251_ MAN-BSA; •. density. elsewhere (Ajioka and Kaplan. 1987), this reaction was both DAB and H20 2 dependent. Although there was an increase in density of a-M· !2~I_ T, the distribution and absolute amounts of alkaline phosphodiesterase and iJ-N-acetylhexoseamini­dase were unaltered (data not shown). This result demon­strates that the endosome is a unique compartment independ­ent of lysosomes and plasma membrane, and that MAN-BSA and a-M· T are internalized into the same compartment. The distribution of radioactivity after the density shift revealed that a significant amount (approximately 25%) of ligand remained at the original density. The concentration of ligands employed was such that 50-70% of all surface recep­tors should have been occupied. The above results could indicate that endosomes may be enriched in one or the other ligand, or, alternatively, it could reflect some damage to endosomes or some other inherent limitation of the technique. To distinguish between these possibilities, cells were incu­bated with either horseradish peroxidase and a-M· l~5I_T or horseradish peroxidase and 125I-MAN-BSA and using the protocol outlined above were subjected to the density shift. As demonstrated in Fig. 2, C and D, the same percentage of 125I-MAN-BSA was shifted as a_M· 125I·T. Since horseradish peroxidase and 125I-MAN·BSA occupy the mannose-terminal glycoprotein receptor (the difference in scale in Fig. 2, C and D, is ascribed to horseradish peroxidase and 12~I-MAN-BSA competing for the same receptor) this result suggests that there are not separate endosomal populations. Rather, the unshifted population reflects some limitation in the system. i.e. damage to the endosome population during preparation. Dissociation of Internalized Receptor-Ligand Complexes­Once receptor-ligand complexes are internalized. the follow­ing two steps are required for proteolytic digestion of the ligand: first, transfer of the ligand from the endosome to the lysosome, and second, proteolytic digestion within the lyso­some. Extended incubation of cells with radiolabeled ligand results in the generation of acid-soluble radioactivity, and the rate of generation of acid-soluble radioactivity exhibits first order kinetics (Kaplan and Nielson. 1979b). To determine whether different ligands enter the lysosome at similar or dissimilar rates, we measured the lag time prior to initiation of degradation. It has been shown that different ligands have characteristic sensitivities to proteolytic enzymes, but if liganda internalized at the same time move to the lysosome at the same rate, the lag time prior to degradation should be similar. To test this hypothesis cells were incubated for 2 min at 37 ·C or for 4 min at 28°C with either a_M· 125I_T or 125I-MAN-BSA. The cells were placed at 0 ·C, and surface-bound ligand was re­moved. The cells were then incubated at the original temper­ature, and the amount of acid-soluble radioactivity in cells and in the media was measured as a function of time. To examine the possibility of bulk transfer of vesicular compo­nents, we also measured the lag time prior to catabolism of a fluid-phase molecule. Cells were incubated with 12~I-MAL- 38 Intracellular Trafficking in Alcf'oiar .Hacrophages BSA in the presence of a high concentration of nonradioactive ~(AL·BSA. The concentration of ~lAL·BSA \ 1 x ~O-' \1) was such that the majority of the receptors were occupied with the nonradioactive ligand. This was ascertamed bv demon· strating that further increases m the concentratIOn or' non­radioactlve .\'lAL-BSA did not affect ceil·assoclated radioae· m·ity. Thus. uptake of the radioactive molecule occurred through tluld-phase pmocytosiS rather than dilution uf the speculc actlvitv of a receptor·bound ligand. At each time pomt the amount of aCid-soluble radioactlv\tv in t he ceil was less than tOe;, at that m the media .. \s t'xoected. the rate of appearance 01 acid-soluble del;radatlon product,; ror eacn li­gand was different at both :37 I data not ;;hown 1 and 26 T 1 Fig. 31. Extrapoiatlon at the appearance uf ac:d-,.;olubie ra· dioactlvlty to the ordmate revealed that degradation 01 each ligand was mltiated at approxImately the same time 1 13.:2 mm for '-'[-~(AN·BSA. 1'2.9 mm tor Lt·~f ;·[·T. and L!.) min tor 12S[·.\'lAL-BSAJ. These values were determlOed bv linear regression analysis and had correlation coeffiCIents' ramnng from 0.96 [Q 0.97. These resuits suggest deliver" 01 all ligand to the lysosomes. whether ()rigmaily receptor bound or not. occurs at the same time. This IS consistent WIth ,)ur demon­stration that fluid-phase markers are located 10 endosomes which contain receptor·ligand complexes I Fig. 21. [f there are receptor·speclfic rate" or' movement. these 40~------------------------------------~ 30 c» :a :l '0 III 20 c( (..J. #. 10 o 10 20 30 JO 50 cO Time (minutes) FIG. 3. [)eg'radation of ligand internalized by nonspecific or receptor-mediated endocytosis at 28°C .. l..lveolar macro· phages were mcubated With different ligands 1:~;r·:\IA~-BSA. <.1:·:\1· 'l~[·T. and il~I-:vtAL· BSA for a l1uid-phase marker lfl the presence of saturatmg amounts of nonradioactive ~IAL·BSA) tor 4 mm at 28 'C. Cells were placed at 0 'C, surface·bound ligand removed. and samples returned to 28 'C. At speCified times media samples were removed and trichloroacetic aCid t TCA l-"oiuble radioact\vltv determmed. At the end of the experiment. the amount of cell.assocl~ted radioactivity waa determined. The data are presented In terms of percent total celio aMOCiated radioactiVity: C. \Z5I-MA~-BSA; .:... '~'I·:\1AL·B:5A; •. <.1:­M · illl (·T. unique rates rnav have different temperature senS!tlVlues. Thus. bv 10werlOg the temperature we :;hould be able to exaggerate such differences. We therefore measured the ap­pearance of aCld-~olubje radioactiVIty produced bv cells inCU­bated at different temperatures. We found that at each tem­perature the rate of catabolIsm was different for each iig-and. while the lag tlme prior to initiation ofdegradatlOn was al:"avs the ,;ame ,Fig' -+1. These results :,upport toe tdea that tne contents ,d \ e ... cies are transported en masse to the Ivsosome. JII)Lemel1( 'ff Receptors tn tne Cell ;)urface-The ;·OilOWIO'; expeClments wpre desu.med to determine It dirIerent receptors. <Jnce tnternalized. are externaltzed at the ~ame ur different rates .. \ potential problem IS that the presence 0{ ~urface receptors con:itltutes a hu!h "background" limiting I)ur aodity ro measure t he rate ot return of receptors tram IOternal poois. We rook advantage o[ the observation that the rate of inter­nalizatIOn or occupIed receptors for ct-:Vl· T and :VlA's·BSA is greater than the rate of internalizatIOn of unoccupIed recep­, nrs. [n cell" exposed to these ligands there 15 a tlme-depend­ent and reverSible decrease 10 the number of surface receptors I Kaplan et ai .. 19I3;,1) Cells were incubated at .37 'C with saturatina: amounts ot L'- :vI· Tor mannan I a molecule which interacts w'Ith the ~lTG receptor) for 90 min to "dnve" occupied receptors inside and decrease the number of surface receptors. Cells were placed at I) 'C and surface-bound ligand removed. Our previous stud· ies demonstrated that the ligand-stripping procedures re­moved greater than 95";; of receptor· bound ligand i Kapian and 'sielson. 1979b: Buys et ai.. 1987), The ceils were then placed at 28 'C to allow for receptor externalization and at speCified times surface binding measured. We chose to do this experiment at 28C to slow down the rate of receptor move­ment to tncrease Gur temporal resolution. The rate of appear­ance ,)1' the Lt·~l· T and ~lTG receptors was SImilar wlth a t, , of 2 mm I Fig-. ,j I. [n three separate experiments there was no sigmticant difference 10 the rates of reappearance of the two receprors. Therefore. in contrast to the signtficant differ­ence in the rate ot internalization of these receptors' their rate of externalization IS the same. To further test our hypothesis. we utilized a different ap­proach. \\'e rirst lOactivated surface receptors and then meas­ured the rate of appearance ot receptors recruited from the internal pool back to the cell surrace. To accomplish this we 80 80 (B) 60 60 40 40 I6j 20 20 ... !... 0 0 5 10 15 20 0 10 15 20 25 fI) C ~IOO (C) 40 (0) ?1 l' 7' 30 ,,0 50 /'." ,er. 20 Z, .-#1' 10 .. 0 12 24 3e 4S 10 0 IS 31 '4 12 90 MINUTES FIG . ..\. Effect of temperature on the rates of dissociation and degradation of internalized ligands. Cells were placed at i) 'C for tiO mIn With different ligands. washed. and then placed at either 37 'C IA l. 34 I B L 28 'C lel. or 20 'C rD L At :lpeclfied times :lam pies of media were removed and trichloroacetic aCId I TCA.)­soluble radioactlvlty measured as described under ~Experlmental Procedures." O.·~5I·MA~·BSA; •. a·M. '2~I·T. 39 8168 Intracellular Trafficking in ALLpIJiar .\facmphm!t''i !O~--------------------~ 08 ; 06 ~ 04 i 02 !,4"'Yle!l FIG, Rate of movement of receptors from the internal pool to the cell surface. \..'t';l~ '.\ere .ncubated ilt:~ (- rq T.in :n the pre,ence 'II ~aturatln" am0unts ,):.·\IT and :nanndn '-etls were men piaced Jt .) 'C'. wasned with EDT."\"conHlln:ng flurrer,; ell remove .;urlace·hOtlnd liil:ana, and placea baCK at ~.~ C:n the ,lGSenc'e or lIgand, . ..\.t speCified urnes .;amp:es were remo\ed, -It C ami i)lndlni rletermlneci ~")r . I, \!A\ B;-;A md, \1 '.' at I) C, in,,,:· :"oeclti, blncim" ''\'-a:; Icaih a" a lunctlOn 1]1 um~ 8. speclr:c ,pecliic binding Jt eqUilibrium_ M,nUles -eml[l)ganthm­at time .; B.n .. c: FIG, 6, Movement of receptors to the surface of cells that had been exposed to trypsin at o°c. Cells were treated wnh tnrpsm as described under "Experimental Procedures," The ceil:; were t hen placed at 28 'C emd at specnied times samples were removed. placed at I) 'C. and surface !}!ndm~ determined tOt -"[·\1A\'·8SA IC) and :'51 ·diiemc transferrInI FH, i 101. Specllic bmdmg was piotted as In Fig. )_ FIG. -:- Effect of trYP8in on the rate of release or internal· ized lZIlI·direrric transferrin. Cells were Incubated at 37 'C WIth l;sl-diferrlc transfemn for 60 min. The cells were placed at I) 'C and surface-bound ligand was removed usmg stripping solutlons as de­SCribed under "Experimental Procedures," An aliquot of cells was exposed to trypsin at 0 'C. and then after neutralizatIOn both the treated and control sample were placed at 28 'C. At specltic tImes aliquots were taken. surface-bound ligand removed. and ceil-aSSOCI' ated radioactivity determmed. C. rate of disappearance of radioactiv­ity trom control ceils: •. from trypsm-treated cells, exposed cells to trypsin at 0 ·C. Cnder these conditions the binding activity of the diferric transferrin and MTG receptors are inactivated! Lamb et at .. 1983: Stahl et at., 1980), CeUs treated with trypsin at O·C were then placed at 28 'C to observe the rate of appearance of surface receptor activity. The rate of appearance of the diferric transferrin or MTG receptor was identical (Fig. 6). The possibility was considered that trypsin treatment may alter membrane recycling and obscure differences in rates. This seems unlikely since t1"ypsm treatment has no effect on a number of responses of macro· phages !DeludIng the i:1Crea"e 'l! .;unaee recepwrs In re"ponse to n"llo-I)Smotlc expu~ure and the tnternailZdtlOn .,( :liiar:d dt a ~peeilic rate I :--;()\,al< t't ~:j"''''1 The receptors (Or ·jlIernc transfernn and \lTC-; are tr\llsm ~en';ltl\e. the ".\! -T recep­tor 1:5 not dnd can ~heretore ;;ene as a (I)ntrol tu measure d~e effects (Jt tr\llsm nn'ourmal" receptor· medIated endoC\1:0S1S. The rate ,11 reled~e l)t Internalized !-diferr:c tran"lernn was -imtiar to control cells and 1n celis expn,ed to tnllsm . Fig. -, The ,·an rhat smail amOU:1ts f)t radlvacuVltv remamed cell associdted atter ; mm ma\' ~er1eC[ the continued recvcim~ 0r' dir'ernc ~ranS[errlO-receptor cumplexes The haif-tlme tor the rate 1)1 receptor reappearance nn the cell ~urface usmg