Calcitonin/Amylin Receptors and Ramps

Our understanding of G protein-coupled receptor function has recently expanded to encompass novel protein interactions that underlie both cell surface receptor expression and the exhibited phenotype. The most notable examples are those involving receptor activity modifying proteins (RAMPs). RAMP association with the calcitonin receptor-like receptor (CRLR) traffics this receptor to the cell surface, where individual RAMPs dictate the expression of unique phenotypes

Exploring Ligand Binding to Calcitonin Gene-Related Peptide Receptors

Class B1 G protein-coupled receptors (GPCRs) are important targets for many diseases, including cancer, diabetes, and heart disease. All the approved drugs for this receptor family are peptides that mimic the endogenous activating hormones. An understanding of how agonists bind and activate class B1 GPCRs is fundamental for the development of therapeutic small molecules. We combined supervised molecular dynamics (SuMD) and classic molecular dynamics (cMD) simulations to study the binding of the calcitonin gene-related peptide (CGRP) to the CGRP receptor (CGRPR). We also evaluated the association and dissociation of the antagonist telcagepant from the extracellular domain (ECD) of CGRPR and the water network perturbation upon binding. This study, which represents the first example of dynamic docking of a class B1 GPCR peptide, delivers insights on several aspects of ligand binding to CGRPR, expanding understanding of the role of the ECD and the receptor-activity modifying protein 1 (RAMP1) on agonist selectivity

Bitopic binding mode of an M1 muscarinic acetylcholine receptor agonist associated with adverse clinical trial outcomes

The realisation of the therapeutic potential of targeting the M1 muscarinic acetylcholine receptor (M1 mAChR) for the treatment of cognitive decline in Alzheimer's disease has prompted the discovery of M1 mAChR ligands showing efficacy in alleviating cognitive dysfunction in both rodents and humans. Among these is GSK1034702, described previously as a potent M1 receptor allosteric agonist, which showed pro-cognitive effects in rodents and improved immediate memory in a clinical nicotine withdrawal test but induced significant side-effects. Here we provide evidence using ligand binding, chemical biology and functional assays to establish that rather than the allosteric mechanism claimed, GSK1034702 interacts in a bitopic manner at the M1 mAChR such that it can concomitantly span both the orthosteric and an allosteric binding site. The bitopic nature of GSK1034702 together with the intrinsic agonist activity and a lack of muscarinic receptor subtype selectivity reported here, all likely contribute to the adverse effects of this molecule in clinical trials. We conclude that these properties, whilst imparting beneficial effects on learning and memory, are undesirable in a clinical candidate due to the likelihood of adverse side effects. Rather, our data supports the notion that "pure" positive allosteric modulators showing selectivity for the M1 mAChR with low levels of intrinsic activity would be preferable to provide clinical efficacy with low adverse responses

Biased allosteric modulation at the CaS receptor engendered by structurally diverse calcimimetics

Background and Purpose Clinical use of cinacalcet in hyperparathyroidism is complicated by its tendency to induce hypocalcaemia, arising partly from activation of calcium-sensing receptors (CaS receptors) in the thyroid and stimulation of calcitonin release. CaS receptor allosteric modulators that selectively bias signalling towards pathways that mediate desired effects [e.g. parathyroid hormone (PTH) suppression] rather than those mediating undesirable effects (e.g. elevated serum calcitonin), may offer better therapies. Experimental Approach We characterized the ligand-biased profile of novel calcimimetics in HEK293 cells stably expressing human CaS receptors, by monitoring intracellular calcium (Ca2+i) mobilization, inositol phosphate (IP)1 accumulation, ERK1/2 phosphorylation (pERK1/2) and receptor expression. Key Results Phenylalkylamine calcimimetics were biased towards allosteric modulation of Ca2+i mobilization and IP1 accumulation. S,R-calcimimetic B was biased only towards IP1 accumulation. R,R-calcimimetic B and AC-265347 were biased towards IP1 accumulation and pERK1/2. Nor-calcimimetic B was unbiased. In contrast to phenylalkylamines and calcimimetic B analogues, AC-265347 did not promote trafficking of a loss-of-expression, naturally occurring, CaS receptor mutation (G670E). Conclusions and Implications The ability of R,R-calcimimetic B and AC-265347 to bias signalling towards pERK1/2 and IP1 accumulation may explain their suppression of PTH levels in vivo at concentrations that have no effect on serum calcitonin levels. The demonstration that AC-265347 promotes CaS receptor receptor signalling, but not trafficking reveals a novel profile of ligand-biased modulation at CaS receptors The identification of allosteric modulators that bias CaS receptor signalling towards distinct intracellular pathways provides an opportunity to develop desirable biased signalling profiles in vivo for mediating selective physiological responses

4-Phenylpyridin-2-one derivatives: a novel class of positive allosteric modulator of the M1 muscarinic acetylcholine receptor

Positive allosteric modulators (PAMs) of the M1 muscarinic acetylcholine receptor (M1 mAChR) are a promising strategy for the treatment of the cognitive deficits associated with diseases including Alzheimer’s and schizophrenia. Herein, we report the design, synthesis, and characterization of a novel family of M1 mAChR PAMs. The most active compounds of the 4-phenylpyridin-2-one series exhibited comparable binding affinity to the reference compound, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (BQCA) (1), but markedly improved positive cooperativity with acetylcholine, and retained exquisite selectivity for the M1 mAChR. Furthermore, our pharmacological characterization revealed ligands with a diverse range of activities, including modulators that displayed both high intrinsic efficacy and PAM activity, those that showed no detectable agonism but robust PAM activity and ligands that displayed robust allosteric agonism but little modulatory activity. Thus, the 4-phenylpyridin-2-one scaffold offers an attractive starting point for further lead optimization

Expression von Parathormone-related Peptide in koronaren Endothelzellen und der glatten Gefäßmuskulatur

Das Parathormon related peptide (PTHrP) wird im gesamten Gefäßsystem einschließlich der koronaren Endothelzellen exprimiert. Im Gegensatz zu den benachbarten Glattmuskelzellen, welche bislang hinsichtlich PTHrP eingehend untersucht wurden, hat man die koronaren Endothelzellen nur als Produktionsstätte des Ventrikels angesehen. Aus diesem Grund blieben bisher die PTHrP-Expression und seine biologische Rolle in diesen Zellen ungeklärt. Deshalb habe ich mich dafür interessiert, ob eine Stimulation a-adrenerger bzw. AT-Rezeptoren die Expression in den koronaren Endothelzellen steigert und ob das endogen exprimierte PTHrP einen sogenannten intrakrinen Effekt in diesem Zelltyp ausübt. Besonders interessant ist diese Fragestellung vor dem Hintergrund, daß diese Zellen keine klassischen Zielzellen des Peptidhormones darstellen, da sie nicht den korrespondierenden PTHrP-Rezeptor besitzen, wie das z. B. bei den Glattmuskelzellen der Fall ist. Was anhand der Ergebnisse gezeigt werden konnte ist die Tatsache, daß durch Stimulation der Zellen mit der a-adrenergen Substanz Phenylephrin im Vergleich zur Stimulation mit Angiotensin II, eine deutliche Steigerung in der Expression des PTHrP hervorgerufen werden konnte. Dieser Effekt konnte jedoch nicht im Stadium der Konfluenz reproduziert werden. Im Gegensatz dazu erhielten wir in den Glattmuskelzellen ein genau umgekehrtes Bild, da hier nur Angiotensin II eine signifikante Expressionssteigerung hervorrief. Deshalb wollte ich mehr über die Lokalisation des endogenen PTHrP in den verschiedenen Proliferationsstadien mit Hilfe der Immunfluoreszenz in Erfahrung bringen. Die Ergebnisse zeigten, daß der Anteil an PTHrP, welcher in den Kern transloziert wird, umso größer ist, je mehr sich die Zellen dem Konfluenzstadium nähern. Diese Tatsache war für mich Anlaß zu vermuten, daß das Peptidhormon einen intrakrinen Effekt auf die koronaren Endothelzellen ausübt. Mit Hilfe der Transfektion der Zellen mit Antisense-Oligonukleotiden gegen PTHrP und der daraus sich ergebenden Herabregulierung seiner Expression untersuchte ich seinen möglichen Einfluß auf die Proliferation bzw. Apoptose. Im Bezug auf die Proliferation ergaben sich keine Hinweise auf eine Beeinflussung. Bei der Betrachtung der Endothelzellen in den unterschiedlichen Konfluenzstadien konnte mit Hilfe einer UV-Bestrahlung in Zellen, in denen der Anteil des nukleären PTHrP normalerweise erhöht wäre, eine stärkere Apoptoserate als unter Basalbedingungen hervorgerufen werden. Im Gegensatz dazu wurde durch eine erhöhte Expression des PTHrP - welche durch Phenylephrin vermittelte wurde -der Anteil der apoptotischen Zellen sowohl gegenüber Basalbedingungen als auch unter UV-Induktion deutlich vermindert. Zusammenfassend läßt sich anhand der Ergebnisse der vorliegenden Studie sagen, daß in koronaren Endothelzellen die PTHrP-Expression durch eine a-Adrenozeptor-Stimulation in einer Zellzyklus-abhängigen und Zelltyp-spezifischen Art und Weise reguliert wird. Diese Studie zeigt eine neue biologische Rolle des PTHrP im Gefäßbett auf, da über den nachgewiesenen intrakrinen Effekt das Peptidhormon einen Anteil am Schutzmechanismus der Endothelschicht vor Apoptose besitzt.Parathyroid hormone related peptide (PTHrP) is expressed throughout the vascular system including coronary endothelial cells. The regulation of endothelial PTHrP expression and the role of PTHrP expression in endothelial cells is not clear. The present study investigates the question whether stimulation of a-adrenergic or angiotensin II receptors increases endothelial expression of PTHrP and whether endogenously expressed PTHrP exerts intracrine effects in coronary endothelial cells. It was found that stimulation of alpha1A-adrenoceptors, but not that of angiotensin II, increases cellular expression of PTHrP in growing, but not in growth arrested, coronary endothelial cells. Angiotensin II increases the expression of PTHrP in smooth muscle cells, but not in endothelial cells. PTHrP enters the nucleus of endothelial cells at the stadium of confluence. This suggests an intracrine effect of PTHrP. It was further investigated whether downregulation of endogenous PTHrP expression by transfection with antisense oligonucleotides alters cell proliferation or apoptosis resistance in growing or non-growing endothelial cells. Downregulation of PTHrP did not modify cell proliferation but increased the amount of UV-induced apoptosis. An increased expression of PTHrP in cells pre-treated with an a-adrenoceptor agonist reduced basal rate of apoptosis and improved resistance against UV-induced apoptosis. These results indicate a novel intracrine effect of PTHrP in coronary endothelial cells that improves cell survival. In endothelial cells, the expression of PTHrP is regulated by alpha-adrenoceptor stimulation in a cell-cycle dependent and cell-type specific manner

Structural and functional diversity among agonist-bound states of the GLP-1 receptor

Recent advances in G-protein-coupled receptor (GPCR) structural elucidation have strengthened previous hypotheses that multidimensional signal propagation mediated by these receptors depends, in part, on their conformational mobility; however, the relationship between receptor function and static structures is inherently uncertain. Here, we examine the contribution of peptide agonist conformational plasticity to activation of the glucagon-like peptide 1 receptor (GLP-1R), an important clinical target. We use variants of the peptides GLP-1 and exendin-4 (Ex4) to explore the interplay between helical propensity near the agonist N terminus and the ability to bind to and activate the receptor. Cryo-EM analysis of a complex involving an Ex4 analog, the GLP-1R and Gs heterotrimer revealed two receptor conformers with distinct modes of peptide-receptor engagement. Our functional and structural data, along with molecular dynamics (MD) simulations, suggest that receptor conformational dynamics associated with flexibility of the peptide N-terminal activation domain may be a key determinant of agonist efficacy.</p

Understanding VPAC receptor family peptide binding and selectivity

The vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) receptors are key regulators of neurological processes. Despite recent structural data, a comprehensive understanding of peptide binding and selectivity among different subfamily receptors is lacking. Here, we determine structures of active, Gs-coupled, VIP-VPAC1R, PACAP27-VPAC1R, and PACAP27-PAC1R complexes. Cryo-EM structural analyses and molecular dynamics simulations (MDSs) reveal fewer stable interactions between VPAC1R and VIP than for PACAP27, more extensive dynamics of VIP interaction with extracellular loop 3, and receptor-dependent differences in interactions of conserved N-terminal peptide residues with the receptor core. MD of VIP modelled into PAC1R predicts more transient VIP-PAC1R interactions in the receptor core, compared to VIP-VPAC1R, which may underlie the selectivity of VIP for VPAC1R over PAC1R. Collectively, our work improves molecular understanding of peptide engagement with the PAC1R and VPAC1R that may benefit the development of novel selective agonists