Ca2؉ and other divalent cations modulate parathyroid hormone secretion by interacting with cell-surface Ca 2؉ -sensing receptors (CaRs). We assessed the ability of these receptors to couple to Ca 2؉ mobilization, inositol phosphate (InsP) accumulation, and cyclic AMP production in different expression systems. In Xenopus laevis oocytes injected with bovine parathyroid CaR cRNA, the addition of extracellular cations to 1.5 mM Ca

Structural determinants within the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor that mediate G-protein activation of adenylate cyclase and phospholipase C are unknown. We investigated the role of the N-terminal region of the third intracellular loop of the opossum PTH/PTHrP receptor in coupling to two signal transduction pathways. We mutated residues in this region by tandem-alanine scanning and expressed these mutant receptors in COS-7 cells and/ The parathyroid hormone (PTH) 1 /PTH-related peptide (PTHrP) receptor is a G-protein-coupled receptor (GPCR) that initiates biological responses to PTH, the major endocrine regulator of calcium and skeletal homeostasis, as well as PTHrP, a cytokine essential for normal endochondral bone formation (1-3). The PTH/PTHrP receptor, by primary sequence alignment, belongs to a distinct subfamily of GPCRs, the PTH/ secretin receptor subfamily, which includes receptors for calcitonin, secretin, vasoactive intestinal polypeptide, gastric inhibitory peptide, glucagon, glucagon-like peptide-1 (GLP-1), corticotrophin-releasing factor, pituitary adenylate cyclase-activating polypeptide (PACAP), and the insect diuretic hormone (4). The PTH/PTHrP receptor mediates its actions by coupling to second messenger generation (cAMP, inositol trisphosphate (IP 3 ), and intracellular Ca 2ϩ ), presumably through activation of G s and G q /G q -like proteins (5, 6). The ability to generate second messengers through two signaling pathways is a distinct functional property shared among many members of the PTH/secretin receptor subfamily. Thus far, the structural features responsible for coupling to dual signaling pathways are unknown for any receptor in this subfamily.Structure-function studies of signal transduction properties of GPCRs have been best described for members of the rhodopsin/adrenergic receptor subfamily. Overall, these studies have showed that all intracellular regions of the receptors contribute to the domains which couple to signaling pathways (7,8). Four intracellular regions (the N-and C-terminal portions of the third intracellular (IC-3) loop, the C-terminal portion of the IC-2 loop, and N-terminal portion of the cytoplasmic tail) have all been shown to play a critical role in G-protein coupling (7,8). Direct sequence comparisons of these intracellular regions between members of the PTH/secretin and the rhodopsin/adrenergic receptor subfamilies, however, have revealed no sequence homologies in these regions. This, therefore, excludes the possibility of identifying shared sequence motifs required for G-protein coupling among these receptors.Studies of signal transduction mediated by the PTH/secretin receptor subfamily have, thus far, provided limited information. Studies comparing different splice variants of members of this subfamily suggested that inserted sequences in the Nterminal region of the IC-1 loop and the C-terminal region of the IC-3 loop could play a role in modulating receptor-G-protein coupling. Insertions in the IC-1 domain of receptors for both...

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.