Amino Acids in the Second and Third Intracellular Loops of the Parathyroid Ca2+-sensing Receptor Mediate Efficient Coupling to Phospholipase C

Abstract

To determine the role of amino acids in the second and third intracellular (IC) loops of the Ca 2؉ -sensing receptor (CaR) in phospholipase C (PLC) activation, we mutated residues in these loops either singly or in tandem to Ala and assessed PLC activity by measuring high extracellular [ (7), and a large group of pheromone receptors (8, 9) and, thus, constitute the family 3 of GPCRs (10). Receptors in the CaR/mGluR subfamily share several structural features. These include a large extracellular amino-terminal domain, seven membrane-spanning regions, three IC loops, and a large cytoplasmic tail (see Fig. 1a). The extracellular domains of CaRs and mGluRs are known to be critical for ligand recognition (11-13). Naturally occurring mutants of the CaR, implicated in the pathogenesis of abnormal Ca 2ϩ -sensing in vivo, occur predominantly in the large amino-terminal domain of this receptor (14,15). Point mutations in this domain, responsible for either gain-of-function or loss-of-function, indicate its key role in the Ca 2ϩ -sensing function of the receptor. IC domains of receptors in the CaR/mGluR subfamily are likely, by analogy to other GPCRs, to be responsible for coupling to G-protein-mediated signal transduction (10,16,17). A comparison of CaRs with the mGluR 1-8 indicates limited sequence conservation in their second IC loops (Ͻ10%) but striking conservation (67 to 85%) in their third IC loops (see Fig. 1b). This observation suggests these latter regions likely share similar function.Mutagenesis of mGluR1 and R5 previously demonstrated that specific residues in IC loops 2 and 3 contribute to PLC activation, whereas other residues were involved in the regulation of cyclic AMP formation (5). Domains of comparable functional significance in the CaR have, to date, not been identified. Studies of kindred with familial benign hypercalcemia and neonatal severe hyperparathyroidism indicated that a CaR with a mutation at residue 795 (R795W) in the aminoterminal portion of the third IC loop had a reduced ability to mobilize intracellular Ca 2ϩ (18). The remaining residues within the second and third IC loops have not been carefully examined. In these studies, we mutated amino acids in IC loops 2 and 3 of the bovine CaR to identify the positions of key signaling residues and structural requirements at those sites. Phe-707 in the second IC loop and 2 residues in the third IC loop, Leu-798 and Phe-802, proved critical to the activation of PLC. Glu-804 proved essential for efficient cell surface expression of CaRs. This work supports the presence of several functional determinants in IC loops 2 and 3 in the stimulation of PLC by and expression of CaRs in mammalian cells.

Molecular Structure and Physiological Functions of GABABReceptors

“…Mutational analysis confirmed the functional importance of conserved residues (K586, M587, and K590) in the i2 loop of GABA B (2) and indicates that the GABA B(1) i2 loop lacks the requirements for interaction with G proteins (97,280). Mutation of K686, a basic residue in the i3 loop of GABA B(2) that plays a critical role in G protein coupling of mGlu1 and CaS receptors (66,102), suppresses functional coupling to G proteins in HEK293 and cerebellar granule cells, corroborating a similar role for this residue (97).…”

Calcium sensing receptor forms complex with and is up-regulated by caveolin-1 in cultured human osteosarcoma (Saos-2) cells

“…CaSR is also related with PLC. Chang and co-workers (Chang et al, 2000) have reported that second and third intracellular loops of the CaSR is coupled with PLC. CaSR belongs to the type III family of G-proteincoupled receptors, which comprises the metabotropic glutamate receptor and putative vomeronasal organ receptors (Brown et al, 1993).…”

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

“…A mutation (R866X) that truncates the C-terminal region after S865 (red) was also generated. All four mutants have been reported previously to impair PI-PLC signalling [17,19]. (B) Effects of iL point mutations and a C-terminus truncation mutant on CaSR total and surface expression.…”

“…In the present study, we set out to investigate whether it is possible to define distinct subsets of molecular requirements for the Ca 2+ o -stimulated activation of G q/11 , G i/o and ERK 1/2 phosphorylation, taking advantage of previous work on the molecular requirements of Ca 2+ o -stimulated PI-PLC activation by the bovine CaSR [17], which exhibits 93% amino acid identity with the human CaSR (hCaSR). This work identified the conserved residue F707 (hCaSR residue F706) in iL-2, and three mutants homologous to hCaSR L797A, F801A and E803A, all of which exhibited marked attenuations in their Ca 2+ ostimulated PI-PLC responses [17].…”

“…This work identified the conserved residue F707 (hCaSR residue F706) in iL-2, and three mutants homologous to hCaSR L797A, F801A and E803A, all of which exhibited marked attenuations in their Ca 2+ ostimulated PI-PLC responses [17]. Furthermore, truncation of the proximal C-terminus beyond hCaSR residue S865, by the introduction of a premature stop codon R866X, abolished PI-PLC signalling [18,19].…”