Christophe Petrel

Delineating a CA2+ binding pocket within the venus flytrap module of the human calcium sensing receptor

The Ca2+-sensing receptor (CaSR) belongs to the class III G-protein-coupled receptors (GPCRs), which include receptors for pheromones, amino acids, sweeteners, and the neurotransmitters glutamate and γ-aminobutyric acid (GABA). These receptors are characterized by a long extracellular amino-terminal domain called a Venus flytrap module (VFTM) containing the ligand binding pocket. To elucidate the molecular determinants implicated in Ca2+ recognition by the CaSR VFTM, we developed a homology model of the human CaSR VFTM from the x-ray structure of the metabotropic glutamate receptor type 1 (mGluR1), and a phylogenetic analysis of 14 class III GPCR VFTMs. We identified critical amino acids delineating a Ca2+ binding pocket predicted to be adjacent to, but distinct from, a cavity reminiscent of the binding site described for amino acids in mGluRs, GABA-B receptor, and GPRC6a. Most interestingly, these Ca2+-contacting residues are well conserved within class III GPCR VFTMs. Our model was validated by mutational and functional analysis, including the characterization of activating and inactivating mutations affecting a single amino acid, Glu-297, located within the proposed Ca2+ binding pocket of the CaSR and associated with autosomal dominant hypocalcemia and familial hypocalciuric hypercalcemia, respectively, genetic diseases characterized by perturbations in Ca2+ homeostasis. Altogether, these data define a Ca2+ binding pocket within the CaSR VFTM that may be conserved in several other class III GPCRs, thereby providing a molecular basis for extracellular Ca2+ sensing by these receptors.

Evidence in Favor of a Calcium-Sensing Receptor in Arterial Endothelial Cells: Studies With Calindol and Calhex 231

Small increases in extracellular Ca2+ dilate isolated blood vessels. In the present study, the possibility that a vascular, extracellular Ca2+-sensing receptor (CaSR) could mediate these vasodilator actions was investigated. Novel ligands that interact with the CaSR were used in microelectrode recordings from rat isolated mesenteric and porcine coronary arteries. The major findings were that (1) raising extracellular Ca2+ or adding calindol, a CaSR agonist, produced concentration-dependent hyperpolarizations of vascular myocytes, actions attenuated by Calhex 231, a negative allosteric modulator of CaSR. (2) Calindol-induced hyperpolarizations were inhibited by the intermediate conductance, Ca2+-sensitive K+ (IKCa) channel inhibitors, TRAM-34, and TRAM-39. (3) The effects of calindol were not observed in the absence of endothelium. (4) CaSR mRNA and protein were present in rat mesenteric arteries and in porcine coronary artery endothelial cells. (5) CaSR and IKCa proteins were restricted to caveolin-poor membrane fractions. We conclude that activation of vascular endothelial CaSRs opens endothelial cell IKCa channels with subsequent myocyte hyperpolarization. The endothelial cell CaSR may have a physiological role in the control of arterial blood pressure.

Calcium sensing receptor-dependent and receptor-independent activation of osteoblast replication and survival by strontium ranelate.

Age‐related osteopenia is characterized by a negative balance between bone resorption and formation. The anti‐osteoporotic drug strontium ranelate was found to reduce bone resorption and to promote bone formation. Here, we investigated the implication of the calcium‐sensing receptor (CaSR) in the response to strontium ranelate using osteoblasts from CaSR knockout [CaSR−/−] and wild‐type [CaSR+/+] mice. We showed that calcium and strontium ranelates increased cell replication in [CaSR−/−] and [CaSR+/+] osteoblasts. Strontium ranelate rapidly increased ERK1/2 phosphorylation in [CaSR+/+] but not in [CaSR−/−] osteoblasts, indicating that strontium ranelate can act independent of the CaSR/ERK1/2 cascade to promote osteoblast replication. We also showed that strontium ranelate prevented cell apoptosis induced by serum deprivation or the pro‐inflammatory cytokines IL‐1β and TNF‐α in [CaSR−/−] and [CaSR+/+] osteoblasts, indicating that CaSR is not the only receptor involved in the protective effect of strontium ranelate on osteoblast apoptosis. Strontium ranelate activated the Akt pro‐survival pathway in [CaSR−/−] and [CaSR+/+] osteoblasts, and pharmacological inhibition of Akt abrogated the anti‐apoptotic effect of strontium ranelate. Furthermore, both the proliferative and anti‐apoptotic effects of strontium ranelate in [CaSR−/−] and [CaSR+/+] osteoblasts were abrogated by selective inhibition of COX‐2. The results provide genetic and biochemical evidence that the effects of strontium ranelate on osteoblast replication and survival involve ERK1/2 and Akt signalling and PGE2 production, independent of CaSR expression. The finding that CaSR‐dependent and CaSR‐independent pathways mediate the beneficial effects of strontium ranelate on osteoblasts, provides novel insight into the mechanism of action of this anti‐osteoporotic agent on osteoblastogenesis.