Selective ligand-induced stabilization of active and desensitized parathyroid hormone type 1 receptor conformations.
ABSTRACT For many G protein-coupled receptors, agonist-induced activation is followed by desensitization, internalization, and resensitization. In most cases, these processes are dependent upon interaction of agonist-occupied receptor with cytoplasmic beta-arrestins. The ligand-induced intramolecular rearrangements of the receptor responsible for the desensitized versus active conformational states, which dictate both the pharmacological properties of ligands and the biological activity of G protein-coupled receptors, have not been fully elucidated. Here, we identify specific interactions between parathyroid hormone (PTH)-related protein and the human PTH type 1 receptor (PTH1Rc) and the related receptor conformational changes that lead to beta-arrestin-2-mediated desensitization. PTH-related protein analogs modified at position 1 induced selective stabilization of the active G protein-coupled state of the receptor, resulting in lack of beta-arrestin-2 recruitment to the cell membrane, sustained cAMP signaling, and absence of ligand-receptor complex internalization. Mechanistically, the ligands modified at position 1, interacting with the extracellular end of helix VI of PTH1Rc, produced a translocation of transmembrane helices V and VI that differed from that induced by the cognate agonist, resulting in significantly different conformations of the third intracellular loop. These results show how specific interactions between PTH1Rc and its ligands may stabilize distinct conformational states, representing either the active G protein-coupled or a desensitized beta-arrestin-coupled receptor state. In addition, they establish that sustained biological activity of PTH1Rc may be induced by appropriately designed agonist ligands.
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ABSTRACT: The PTH type 1 receptor (PTH1R) and PTHrP are expressed in vessels, where they contribute to regulating vascular smooth muscle cell (VSMC) function. Elevated PTHrP levels in VSMC are often associated with hyperplasia. In contrast, exogenous PTHrP, acting through the PTH1R, inhibits VSMC proliferation. In this study, we investigated the regulation of PTH1R expression by endogenous PTHrP and the associated effects on VSMC proliferation. Blocking binding of secreted PTHrP fragments to the PTH1R by treatment with either an antagonist or an antibody against PTHrP, and inhibition of PTHrP expression by small interfering RNA significantly increased PTH1R expression. Interestingly, treatment of the cells with a PTHrP analog (Bpa(1)-PTHrP) that activates the PTH1R without inducing its internalization had the same effect on receptor expression. To examine the association between receptor expression and the antiproliferative effect of N-terminal fragments of PTHrP, VSMC were treated with exogenous PTHrP (1-36) acutely and chronically to induce receptor down-regulation. Stimulation of VSMC with exogenous PTHrP (1-36) significantly reduced cell proliferation during the first 18 h of treatment but was no longer effective after 3 d, a time when PTH1R was down-regulated. In contrast, treatment with the noninternalizing agonist Bpa(1)-PTHrP strongly inhibited cell proliferation at all time points. In conclusion, our study show that PTHrP, after its intracellular processing and secretion, promotes down-regulation of the PTH1R in VSMC, thereby regulating cell proliferation in an auto/paracrine fashion. This regulatory mechanism may have important implication during vascular remodeling, in particular in the development of neointima after arterial injury, where PTHrP overexpression occurs.Molecular Endocrinology 08/2009; 23(10):1681-90. · 4.75 Impact Factor
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ABSTRACT: Cell surface G protein-coupled receptors (GPCRs) drive numerous signaling pathways involved in the regulation of a broad range of physiologic processes. Today, they represent the largest target for modern drugs development with potential application in all clinical fields. Recently, the concept of "ligand-directed trafficking" has led to a conceptual revolution in pharmacological theory, thus opening new avenues for drug discovery. Accordingly, GPCRs do not function as simple on-off switch but rather as filters capable of selecting the activation of specific signals and thus generating texture responses to ligands, a phenomenon often referred to as ligand-biased signaling. Also, one challenging task today remains optimization of pharmacological assays with increased sensitivity so to better appreciate the inherent texture of ligands. However, considering that a single receptor has pleiotropic signaling properties and that each signal can crosstalk at different levels, biased activity remains thus difficult to evaluate. One strategy to overcome these limitations would be examining the initial steps following receptor activation. Even, if some G protein independent functions have been recently described, heterotrimeric G protein activation remains a general hallmark for all GPCRs families and the first cellular event subsequent to agonist binding to the receptor. Herein, we review the different methodologies classically used or recently developed to monitor G protein activation and discussed them in the context of G protein biased-ligands.Current pharmaceutical design 01/2012; 18(2):128-44. · 4.41 Impact Factor
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ABSTRACT: 'Functional selectivity' refers to the ability of a ligand to activate and/or inhibit only a subset of the signals capable of emanating from its cognate G-protein-coupled receptor (GPCR). Whereas conventional GPCR agonism and antagonism can be viewed as modulating the quantity of efficacy, functionally selective or 'biased' ligands qualitatively change the nature of information flow across the plasma membrane, raising the prospect of drugs with improved therapeutic efficacy or reduced side effects. Nonetheless, there is little experimental evidence that biased ligands offer advantages over conventional agonists/antagonists in vivo. Recent work with the type I parathyroid hormone receptor (PTH(1) R) suggests that biased ligands that selectively activate G-protein-independent arrestin-mediated signalling pathways may hold promise in the treatment of osteoporosis. Parathyroid hormone (PTH) is a principle regulator of bone and calcium metabolism. In bone, PTH exerts complex effects; promoting new bone formation through direct actions on osteoblasts while simultaneously stimulating bone loss through indirect activation of osteoclastic bone resorption. Although the conventional PTH(1) R agonist teriparatide, PTH(1-34), is effective in the treatment of osteoporosis, its utility is limited by its bone-resorptive effects and propensity to promote hypercalcaemia/hypercalcuria. In contrast, d-Trp(12) ,Tyr(34) -bPTH(7-34) (PTH-βarr), an arrestin pathway-selective agonist for the PTH(1) R, induces anabolic bone formation independent of classic G-protein-coupled signalling mechanisms. Unlike PTH(1-34), PTH-βarr appears to 'uncouple' the anabolic effects of PTH(1) R activation from its catabolic and calcitropic effects. Such findings offer evidence that arrestin pathway-selective GPCR agonists can elicit potentially beneficial effects in vivo that cannot be achieved using conventional agonist or antagonist ligands.British Journal of Pharmacology 04/2011; 164(1):59-67. · 5.07 Impact Factor