| Recent advances in our understanding of the pluridimensional nature of GPCR signaling have provided new insights into how orthosteric ligands regulate receptors, and how the phenomenon of functional selectivity or ligand 'bias' might be exploited in pharmaceutical design. GPCRs are sensitive to changes in ligand structure which lends the activated GPCR the capacity to dissociate G protein-dependent and arrestin-dependent signaling. In addition to their role in GPCR desensitization and sequestration, arrestins also serve as signaling scaffolds independent of G protein signaling by recruiting regulatory proteins to the desensitized ligand-occupied receptor. Given the nature of GPCR signaling capacity, the characterization ligands efficacy is assay dependent. This dissertation explores the in vitro characterization of functionally selective ligands for the human parathyroid hormone receptor type 1 (hPTH1R) by a multiplex assay approach, which encompasses both G protein and arrestin-mediated processes, to obtain intrinsic relative activity (RA;) profiles for a panel of six PTH analogs. In order to further explore arrestin function, we explored how activation of GPCRs with different structures dictates differential regulation of arrestin function. We correlated conventional ligand mediated activation of a GPCR panel for G protein secondary messenger production, arrestin recruitment and the role of arrestins in ERK1/2 activation with arrestin3 conformational profiles as determined by intramolecular FIAsH-BRET. We then followed up our study by assessing how the chemically discrete panel of hPTH1R ligands differentially regulates arrestin function. By evaluating whether arrestin pathway-selective ligands which do not form stable receptor/arrestin complexes induce receptor desensitization and differences in arrestin3 conformational profiles, we report for the first time that arrestin-dependent signaling can be pharmacologically dissociated from arrestin-mediated hPTH1R desensitization, and expand the range of ligand efficacy that can be obtained using "biased" GPCR ligands. We develop on these results by relating in vitro efficacy to in vivo biological action of functionally selective ligands and how the information obtained from this dissertation and the future work steamed from it will facilitate the promise of rationally designing bias ligands for therapeutic intervention. |
