| Description |
The Hedgehog (Hh) pathway is a cell signaling pathway that is involved in embryonic development and adult tissue maintenance in vertebrates. Improper functioning of this pathway can lead to developmental disorders and several forms of cancer. Despite the pathway's biological importance, the transduction of Hh signal from the membrane to the nucleus remains unclear. Specifically, the biochemical mechanism by which the twelve-pass membrane protein PATCHED1(PTCH1) inhibits G proteincoupled receptor (GPCR) SMOOTHENED (SMO), as well as how SMO communicates to the transcription factor GLI, is still not fully understood in the pathway. My thesis addresses these unclear mechanisms in two parts. The first project involved establishing a novel cell-free system to study PTCH1-SMO inhibition in giant plasma membrane vesicles (GPMVs). The second project addresses the role of G Protein-Coupled Receptor Kinase 2 (GRK2), an essential pathway component involved in the SMO-GLI communication process, by mapping GRK2 phosphorylation sites on the intracellular domain of SMO and testing the functional significance of these residues. Prior research has led to a model in which PTCH1 harnesses the energy from transmembrane Na+ gradients to transport cholesterol, the likely endogenous SMO ligand, away from its binding site in the SMO seven-transmembrane domain[6]. This model was developed largely via experiments in living cells or organisms which cannot directly prove the existing hypothesis biochemically. To circumvent this, I employ GPMVs which create a vesicle with an intact membrane. In this cell free system, if I capture PTCH1 and SMO, and the PTCH1 inhibition of SMO is recapitulated, then this would reveal that the only requirements for this inhibition are the few elements that are contained within the GPMV. Additionally, if such a PTCH1-SMO system were captured, I could test the effect of varying Na+ concentrations in the inner and outer compartments of the vesicles without any secondary effects that would occur in a living cell. I successfully created GPMVs, and, once the purification process is optimized, I will perform experiments that elucidate the role of Na+ gradients on PTCH1 behavior. Downstream in the pathway, GRK2 has been postulated to phosphorylate the intracellular domain of SMO which in turn causes the binding of one or more downstream accessory proteins [2]. However, previous work has shown that when experimentally determined phosphorylation residues for mammalian SMO are mutated there is no effect on Hh activation. This result led to the conclusion that GRK2 kinase activity had another target within the pathway[3]. We postulate that the previous in vitro experiments used to identify these phosphorylation sites may have missed, or misidentified, several crucial phosphorylation sites that occur under physiological conditions in living cells. In the present work, we use liquid chromatography / mass spectroscopy (LC/MS) on purified forms of SMO from mammalian cells to map these crucial GRK2 phosphorylation sites. I then mutated these phosphorylation residues to alanine, a non-phosphorylatable amino acid, and tested the effects on Hh activation in both organismal and cell-based experimental models. These experiments indicated that our mutations caused near-complete elimination of Hh pathway activity in both cell culture and zebrafish embryos. These finding strongly suggest that the phosphorylation of SMO by GRK2 is an essential step for Hh activation. This finding, with results from other members of the Myers lab, shows the phosphorylation of SMO by GRK2 orchestrates the sequestration of PKA at the membrane, and, thus, prevents PKA from inhibiting GLI transcription factors. My experiments have therefore helped to link changes in the SMO activity state to the regulation of GLI transcription factors, leading to a unified model for Hh signal transduction from the membrane to the nucleus. Furthermore, because SMO activation of GLI underlies several cancers, these findings suggest that drugs that interfere with GRK2 kinase activity or GRK2 targeting of SMO may serve as valuable therapeutic agents. |
