Effects of a Calcimimetic Compound and Naturally Activating Mutations on the Human Ca²⁺Receptor and on Ca²⁺Receptor/Metabotropic Glutamate Chimeric Receptors

Abstract

Naturally occurring mutations identified in subjects with autosomal dominant hypocalcemia (ADH) and the calcimimetic compound, R-568, have both been reported to increase Ca2+ sensitivity of the Ca2+ receptor (CaR). To gain insight into their mechanism of action, we studied interactions between four different ADH mutations located in the amino-terminal extracellular domain (ECD) and R-568. We found that R-568 increased the sensitivity of three of the ADH mutant receptors, but the Leu125Pro mutant appeared to be maximally left-shifted in that neither R-568 addition nor combining other ADH mutations with Leu125Pro gave increases in sensitivity comparable to those seen with the three other ADH mutations studied. We also made use of truncation and deletion mutants of the CaR and CaR/metabotropic glutamate receptor type 1 (mGluR1) chimeras to study both the site of action of R-568 and the effect of the Leu125Pro activating mutation. R-568 was effective in receptor constructs containing the seven transmembrane domain (7TM) of the CaR, but not in those containing the mGluR1 7TM. R-568, moreover, imparted Ca2+ responsiveness to CaR constructs lacking all or part of the CaR ECD. The Leu125Pro mutation in contrast conferred no or minimal increase in Ca2+ responsiveness to CaR constructs lacking part of the CaR ECD but showed a striking increase in basal activity in the context of chimeras containing an mGluR1 7TM. Our results localize the site of action of NPS-568 specifically to the CaR 7TM. Our results with the Leu125Pro mutant, furthermore, suggest that the mGluR1 7TM domain may be more permissive for activation than the 7TM domain of the CaR.

Bone and the Kidney: A Systems Biology Approach to the Molecular Mechanisms of Renal Osteodystrophy

“…In parathyroid tissue, they decrease the threshold for CaSR activation by extracellular calcium ions and diminish PTH secretion directly without aggravating disturbances in mineral metabolism that have been associated with adverse clinical outcomes [52]. The calcimimetic agent, NPS-568, has been shown to act specifically on the calcium receptor's 7 transmembrane domain [53].…”

The Role of Cysteines and Charged Amino Acids in Extracellular Loops of the Human Ca2+Receptor in Cell Surface Expression and Receptor Activation Processes

“…The hCaR belongs to the structurally unique family 3 characterized by very large amino-terminal extracellular domains (ECDs) that includes metabotropic glutamate receptors (mGluR1-8), putative pheromone receptors of the vomeronasal organ, sweet taste receptors 1-3, and ␥-aminobutyric acid B receptors. Studies of chimeric receptors containing hCaR ECD and mGluR1 7TM domains have confirmed that hCaR ECD confers responsiveness to [Ca 2ϩ ] o on the chimeric receptors (5,6). This defines a ligand-binding role similar to that defined for the ECD structures of mGluR1 and other family 3 receptors (7).…”

“…An hCaR mutant 7TM construct lacking the ECD (T903-Rhoc) responds to [Ca 2ϩ ] o and other di-and poly-cations (5, 6, 9). Furthermore, (R)-N-(3-methoxy-␣-phenylethyl)-3-(2Ј-chlorophenyl)-1-propylamine hydrochloride (NPS-R568), an allosteric calcimimetic compound, synergistically enhances these cationic responses (5,6,9).An acidic residue (Glu837) located in extracellular loop (EL) 3 of the 7TM has been reported to interact with NPS-R568 but might not be involved in Ca 2ϩ binding (10). We had previously hypothesized that a cluster of acidic residues in…”

“…An hCaR mutant 7TM construct lacking the ECD (T903-Rhoc) responds to [Ca 2ϩ ] o and other di-and poly-cations (5, 6, 9). Furthermore, (R)-N-(3-methoxy-␣-phenylethyl)-3-(2Ј-chlorophenyl)-1-propylamine hydrochloride (NPS-R568), an allosteric calcimimetic compound, synergistically enhances these cationic responses (5,6,9).…”

Role of the calcium-sensing receptor in parathyroid gland physiology

“…It is likely, however, that l-amino acids and phenylalkylamines interact with different portions of the CaSR. Whereas l-amino acids interact with specific loci in the extracellular domain as mentioned previously, calcimimetic agents bind most likely to the membrane-spanning portion of the receptor (39).…”