Cell Surface Calcium-Sensing Receptor Heterodimers: Mutant Gene Dosage Affects Ca2+ Sensing but Not G Protein Interaction.

The calcium-sensing receptor is a homodimeric class C G protein-coupled receptor (GPCR) that senses extracellular Ca2+ (Ca2+ o ) via a dimeric extracellular Venus flytrap (VFT) unit that activates G protein-dependent signaling via twin Cysteine-rich domains linked to transmembrane heptahelical (HH) bundles. It plays a key role in the regulation of human calcium and thus mineral metabolism. However, the nature of interactions between VFT units and HH bundles, and the impacts of heterozygous or homozygous inactivating mutations, which have implications for disorders of calcium metabolism are not yet clearly defined. Herein we generated CaSR-GABAB1 and CaSR-GABAB2 chimeras subject to GABAB -dependent endoplasmic reticulum sorting to traffic mutant heterodimers to the cell surface. Transfected HEK-293 cells were assessed for Ca2+ o -stimulated Ca2+ i mobilization using mutations in either the VFT domains and/or HH bundle intraloop-2 or intraloop-3. When the same mutation was present in both VFT domains of receptor dimers, analogous to homozygous neonatal severe hyperparathyroidism (NSHPT), receptor function was markedly impaired. Mutant heterodimers containing one wild-type (WT) and one mutant VFT domain, however, corresponding to heterozygous familial hypocalciuric hypercalcemia type-1 (FHH-1), supported maximal signaling with reduced Ca2+ o potency. Thus two WT VFT domains were required for normal Ca2+ o potency and there was a pronounced gene-dosage effect. In contrast, a single WT HH bundle was insufficient for maximal signaling and there was no functional difference between heterodimers in which the mutation was present in one or both intraloops; ie, no gene-dosage effect. Finally, we observed that the Ca2+ o -stimulated CaSR operated exclusively via signaling in-trans and not via combined in-trans and in-cis signaling. We consider how receptor asymmetry may support the underlying mechanisms. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Development of Covalent, Clickable Probes for Adenosine A1 and A3 Receptors.

Adenosine receptors are attractive therapeutic targets for multiple conditions, including ischemia-reperfusion injury and neuropathic pain. Adenosine receptor drug discovery efforts would be facilitated by the development of appropriate tools to assist in target validation and direct receptor visualization in different native environments. We report the development of the first bifunctional (chemoreactive and clickable) ligands for the adenosine A1 receptor (A1R) and adenosine A3 receptor (A3R) based on an orthosteric antagonist xanthine-based scaffold and on an existing structure-activity relationship. Bifunctional ligands were functional antagonists with nanomolar affinity and irreversible binding at the A1R and A3R. In-depth pharmacological profiling of these bifunctional ligands showed moderate selectivity over A2A and A2B adenosine receptors. Once bound to the receptor, ligands were successfully "clicked" with a cyanine-5 fluorophore containing the complementary "click" partner, enabling receptor detection. These bifunctional ligands are expected to aid in the understanding of A1R and A3R localization and trafficking in native cells and living systems.

International Union of Basic and Clinical Pharmacology. CVIII. Calcium-Sensing Receptor Nomenclature, Pharmacology, and Function.

The calcium-sensing receptor (CaSR) is a class C G protein-coupled receptor that responds to multiple endogenous agonists and allosteric modulators, including divalent and trivalent cations, L-amino acids, γ-glutamyl peptides, polyamines, polycationic peptides, and protons. The CaSR plays a critical role in extracellular calcium (Ca2+ o) homeostasis, as demonstrated by the many naturally occurring mutations in the CaSR or its signaling partners that cause Ca2+ o homeostasis disorders. However, CaSR tissue expression in mammals is broad and includes tissues unrelated to Ca2+ o homeostasis, in which it, for example, regulates the secretion of digestive hormones, airway constriction, cardiovascular effects, cellular differentiation, and proliferation. Thus, although the CaSR is targeted clinically by the positive allosteric modulators (PAMs) cinacalcet, evocalcet, and etelcalcetide in hyperparathyroidism, it is also a putative therapeutic target in diabetes, asthma, cardiovascular disease, and cancer. The CaSR is somewhat unique in possessing multiple ligand binding sites, including at least five putative sites for the "orthosteric" agonist Ca2+ o, an allosteric site for endogenous L-amino acids, two further allosteric sites for small molecules and the peptide PAM, etelcalcetide, and additional sites for other cations and anions. The CaSR is promiscuous in its G protein-coupling preferences, and signals via Gq/11, Gi/o, potentially G12/13, and even Gs in some cell types. Not surprisingly, the CaSR is subject to biased agonism, in which distinct ligands preferentially stimulate a subset of the CaSR's possible signaling responses, to the exclusion of others. The CaSR thus serves as a model receptor to study natural bias and allostery. SIGNIFICANCE STATEMENT: The calcium-sensing receptor (CaSR) is a complex G protein-coupled receptor that possesses multiple orthosteric and allosteric binding sites, is subject to biased signaling via several different G proteins, and has numerous (patho)physiological roles. Understanding the complexities of CaSR structure, function, and biology will aid future drug discovery efforts seeking to target this receptor for a diversity of diseases. This review summarizes what is known to date regarding key structural, pharmacological, and physiological features of the CaSR.

Roles of intraloops-2 and -3 and the proximal C-terminus in signalling pathway selection from the human calcium-sensing receptor.

The calcium-sensing receptor (CaSR) couples to signalling pathways via intracellular loops 2 and 3, and the C-terminus. However, the requirements for signalling are largely undefined. We investigated the impacts of selected point mutations in iL-2 (F706A) and iL-3 (L797A and E803A), and a truncation of the C-terminus (R866X) on extracellular Ca(2+) (Ca(2+)o)-stimulated phosphatidylinositol-specific phospholipase-C (PI-PLC) and various other signalling responses. CaSR-mediated activation of PI-PLC was markedly attenuated in all four mutants and similar suppressions were observed for Ca(2+)o-stimulated ERK1/2 phosphorylation. Ca(2+)o-stimulated intracellular Ca(2+) (Ca(2+)i) mobilization, however, was relatively preserved for the iL-2 and iL-3 mutants and suppression of adenylyl cyclase was unaffected by either E803A or R866X. The CaSR selects for specific signalling pathways via the proximal C-terminus and key residues in iL-2, iL-3.

Calcium-sensing receptor-dependent activation of CREB phosphorylation in HEK293 cells and human parathyroid cells.

In addition to its acute effects on hormone secretion, epithelial transport, and shape change, the calcium-sensing receptor (CaSR) modulates the expression of genes that control cell survival, proliferation, and differentiation as well as the synthesis of peptide hormones and enzymes. In the present study, we investigated the impacts of a CaSR agonist and several CaSR modulators on phosphorylation of transcription factor CREB residue Ser(133) in CaSR-expressing HEK293 (HEK-CaSR) cells and human adenomatous parathyroid cells. Elevated Ca(2+)o concentration had no effect on CREB phosphorylation (p-CREB) in control HEK293 cells but stimulated p-CREB in both HEK-CaSR cells and human parathyroid cells. In addition, p-CREB was stimulated by the positive modulator cinacalcet and inhibited by the negative modulator NPS 2143 in both CaSR-expressing cell types. Two positive modulators that bind in the receptor's Venus Fly Trap domain, l-phenylalanine and S-methylglutathione, had no effect on p-CREB in HEK-CaSR cells, demonstrating the existence of pronounced signaling bias. Analysis of the signaling pathways using specific inhibitors demonstrated that phosphoinositide-specific phospholipase C and conventional protein kinase C isoforms make major contributions to Ca(2+)o-induced p-CREB in both cell-types, suggesting key roles for Gq/11. In addition, in parathyroid cells but not HEK-CaSR cells, activation of p-CREB was dependent on Gi/o, demonstrating the existence of cell type-specific signaling.