Edited by Ned ManteiKeywords: Calcium-sensing receptor G-protein coupled receptor Biased signalling Phosphatidylinositol-specific phospholipase-C Adenosine 3 0 ,5 0 -cyclic monophosphate ERK 1/2 a b s t r a c tThe 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 phosphatidylinositolspecific 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 ERK 1/2 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.

Negative allosteric modulators (NAMs) of the human calciumsensing receptor (CaSR) have previously failed to show efficacy in human osteoporosis clinical trials, but there is now significant interest in repurposing these drugs for hypocalcemic disorders and inflammatory lung diseases. However, little is known about how CaSR NAMs inhibit the response to endogenous activators. An improved understanding of CaSR negative allosteric modulation may afford the opportunity to develop therapeutically superior CaSR-targeting drugs. In an attempt to elucidate the mechanistic and structural basis of allosteric modulation mediated by the previously reported NAM, calhex231, we herein demonstrate that calhex231 actually potentiates or inhibits the activity of multiple CaSR agonists depending on whether it occupies one or both protomers in a CaSR dimer. These findings reveal a novel mechanism of mode-switching at a Class C G protein-coupled receptor that has implications for drug discovery and potential clinical utility.

The calcium-sensing receptor is a homodimeric class C G protein-coupled receptor (GPCR) that senses extracellular Ca 2+ (Ca 2+ 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-GABA B1 and CaSR-GABA B2 chimeras subject to GABA B -dependent endoplasmic reticulum sorting to traffic mutant heterodimers to the cell surface. Transfected HEK-293 cells were assessed for Ca 2+ o -stimulated Ca 2+ 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 Ca 2+ o potency. Thus two WT VFT domains were required for normal Ca 2+ 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 Ca 2+ 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.

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