The Venus Fly Trap Domain of the Extracellular Ca2+-sensing Receptor Is Required for l-Amino Acid Sensing

Abstract

We previously demonstrated that the human calciumsensing receptor (CaR) is allosterically activated by Lamino acids (Conigrave, A. D., Quinn, S. J., and Brown, E. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4814 -4819). However, the domain-based location of amino acid binding has been uncertain. We now show that the Venus Fly Trap (VFT) domain of CaR, but none of its other major domains, is required for amino acid sensing. Several constructs were informative when expressed in HEK293 cells. First, the wild-type CaR exhibited allosteric activation by L-amino acids as previously observed. Second, two CaR-mGlu chimeric receptor constructs that retained the VFT domain of CaR, one containing the extracellular Cys-rich region of CaR and the other containing the Cys-rich region of the rat metabotropic glutamate type-1 (mGlu-1) receptor, together with the rat mGlu-1 transmembrane region and C-terminal tail, retained amino acid sensing. Third, a CaR lacking residues 1-599 of the N-terminal extracellular head but retaining an intact CaR transmembrane region and a functional but truncated C terminus (headless-T903 CaR) failed to respond to L-amino acids but retained responsiveness to the type-II calcimimetic NPS R-467. Finally, a T903 CaR control that retained an intact N terminus also retained L-amino acid sensing. Taken together, the data indicate that the VFT domain of CaR is necessary for L-amino acid sensing and are consistent with the hypothesis that the VFT domain is the site of L-amino acid binding. The findings support the concept that the mGlu-1 amino acid binding site for L-glutamate is conserved as an L-amino acid binding site in its homolog, the CaR.The extracellular Ca 2ϩ -sensing receptor (CaR) 1 plays a key role in the regulation of whole body calcium metabolism. In keeping with this, the CaR-null mouse exhibits loss of feedback control of parathyroid hormone secretion, hyperparathyroidism, and a metabolic bone disease (2). Furthermore, inactivating and activating mutations of the receptor in humans have been shown to induce various disorders of calcium metabolism (for review, see Ref. 3).Although the CaR is a key molecular regulator of whole body calcium metabolism, it presents something of a conundrum. It is widely expressed in mammalian tissues, including tissues such as the brain, that are not clearly involved in calcium metabolism. Furthermore, its closest relatives in molecular terms are members of sub-group C of the G protein-coupled receptors, receptors for specific amino acids such as L-glutamate (mGlus) and glutamate analogs, e.g. GABA. The large extracellular heads of these receptors are related to nutrientsensing, bacterial periplasmic-binding proteins (4). The finding that the CaR is allosterically activated by a broad spectrum of L-amino acids, including aromatics such as L-Phe and L-Trp and aliphatic and polar amino acids such as L-Ala and L-Ser, has the effect of drawing it closer functionally to other members of subgroup C (1). However, the site of amino acid binding has been unclear. A si...

Calcium-sensing receptor is a physiologic multimodal chemosensor regulating gastric G-cell growth and gastrin secretion

“…CaR, similar to other class 3 family GPCRs that bind amino acids such as the metabotropic glutamate (mGluR 1 ) (23) and heterodimeric GABA B R (24) receptors, shares a functional Nterminal Venus Fly Trap domain. Unlike the mGluR 1 and GABA B R but similar to the taste receptor type 1 members 1 and 3 amino acid sensing taste receptor heterodimer (25) and the less well-characterized nutrient-sensing receptor, GPRC6A (26), CaR recognizes a broad array of L-amino acids interacting within the Venus Fly Trap domain (27). However, CaR has a preference for aromatic amino acids, such as Phe and Trp, in vitro in transfected HEK293 cells (11) and acutely isolated human parathyroid cells (14).…”

“…This acid stimulatory effect of cinacalcet acting on both the G (42) and parietal cell (18) may warrant acid suppressive therapy to prevent cinacalcet intolerance caused by hyperacidity in vulnerable hemodialysis patients on long-term therapy for secondary hyperparathyroidism (43). The inhibition of gastrin secretion by NPS 2143 suggests that clinical trials of calcilytics for the treatment of osteoporosis should monitor potential deleterious inhibition of CaR on nonclassical Ca 2+ regulatory tissues (9,27,28).…”

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

“…The CaSR binds and responds to various endogenous ligands including not only extracellular Ca 2+ (Ca 2+ o ) and Mg 2+ but also organic multivalent cations such as spermine, which acts as an allosteric agonist [8] and L-amino acids, which act as positive modulators (review: [9]) that bind in the receptor's VFT domain [10]. In addition, the CaSR is activated by synthetic modulators (calcimimetics) including the clinically effective phenylalkylamine cinacalcet, which bind in the receptor's heptahelical domain [11].…”

Allosteric Modulation of the Calcium-sensing Receptor by γ-Glutamyl Peptides

“…S1). The results indicate that ␥-glutamyl peptides and S-methylgluta- (14) and glutathione (21) binding sites to the VFT domain and we previously identified T145A/S170T as a double mutant form of the VFT domain with near-normal Ca 2ϩ o -sensitivity but markedly impaired L-amino acid sensing (15). In the current study, we first confirmed that this mutant exhibited normal or near-normal Ca 2ϩ o sensitivity in the absence of L-Phe or S-methylglutathione (Fig.…”

“…1). Based on chimeric receptor and mutational analyses, L-amino acids bind in the receptor N-terminal Venus Fly Trap (VFT) domain (14) and the effects of L-amino acids are selectively impaired by a double mutant (T145A/S170T), which exhibits normal Ca 2ϩ o -sensing (15). Comparative molecular modeling of the mGlu-1 and CaR VFT domain ligand binding surfaces indicates that, whereas the amino acid side-chain binding region is tightly defined by a cluster of positively charged residues in mGlu-1 and other mGlus (16,17) it is relatively unrestricted in the CaR and closely related class C GPCRs including GPRC6A and T1R1 (18).…”