G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters, representing the largest group of therapeutic targets. Recent studies show that some GPCRs signal through both G protein and arrestin pathways in a ligand-specific manner. Ligands that direct signaling through a specific pathway are known as biased ligands. The arginine-vasopressin type 2 receptor (V2R), a prototypical peptide-activated GPCR, is an ideal model system to investigate the structural basis of biased signaling. Although the native hormone arginine-vasopressin leads to activation of both the stimulatory G protein (Gs) for the adenylyl cyclase and arrestin pathways, synthetic ligands exhibit highly biased signaling through either Gs alone or arrestin alone. We used purified V2R stabilized in neutral amphipols and developed fluorescence-based assays to investigate the structural basis of biased signaling for the V2R. Our studies demonstrate that the Gs-biased agonist stabilizes a conformation that is distinct from that stabilized by the arrestin-biased agonists. This study provides unique insights into the structural mechanisms of GPCR activation by biased ligands that may be relevant to the design of pathway-biased drugs.
Earlier, we proposed that the interaction of gizzard calponin with F-actin, promoting the inhibition of the actomyosin ATPase activity, involves the NH2-terminal portion of the calponin segment Ala145-Tyr182 (Mezgueldi, M., Fattoum, A., Derancourt, J., and Kassab, R. (1992) J. Biol. Chem. 267, 15943-15951). In this work, we have directly probed this region for actin binding sites using five peptide analogs covering different stretches of the sequence Thr133-Ile163. Co-sedimentation with F-actin, actomyosin ATPase measurements, and zero-length cross-linking reactions demonstrated that the 19-residue sequence Ala145-Ile163 is essential for actin interaction and ATPase inhibition. Furthermore, each peptide was tested for binding to the Ca(2+)-dependent proteins, caltropin and calmodulin, in both an actomyosin ATPase assay and an affinity chromatographic assay. The results revealed the 11-residue segment Gln153-Ile163, representing the COOH-terminal moiety of the F-actin binding sequence, as a crucial region for the high affinity binding of these regulatory proteins with concomitant removal of the ATPase inhibition. The 153-163 stretch contained also interactive sites for tropomyosin as assessed by affinity chromatography and spectrofluorometry. Collectively, the data support our initial results and highlight the ability of the multifunctional 145-163 region to serve as a potent regulatory domain of the smooth muscle calponin.
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