Evidence for Distinct Cation and Calcimimetic Compound (NPS 568) Recognition Domains in the Transmembrane Regions of the Human Ca2+ Receptor

Kausik Ray,John Northup

doi:10.1074/jbc.m202113200

Abstract

The Ca(2+) receptor, a member of the family 3 of G protein-coupled receptors (GPCR), responds not only to its primary physiological ligand Ca(2+) but also to other di- and trivalent metals (Mg(2+), Gd(3+)) and the organic polycations spermine and poly-l-Arginine. As has been found for other family 3 GPCRs, the large amino-terminal extracellular domain (ECD) of the Ca(2+) receptor is the primary Ca(2+) binding domain. To examine how the signal is propagated from the ECD to the seven-transmembrane core domain (7TM) we constructed a Ca(2+) receptor mutant (T903-Rhoc) lacking the entire ECD but containing the 7TM. We have found that this structure initiates signaling in human embryonic kidney (HEK) 293 cells stably expressing the construct. One or more cation recognition sites are also located within the 7TM. Not only Ca(2+), but also several other Ca(2+) receptor-specific agonists, Mg(2+), Gd(3+), spermine, and poly-l-Arginine, can activate T903-Rhoc truncated receptor-initiated phosphoinositide hydrolysis in HEK 293 cells. The phenylalkylamine compound, NPS 568, identified as a positive allosteric modulator of the Ca(2+) receptor can selectively potentiate the actions of Ca(2+) and other polycationic agonists on the T903-Rhoc receptor. Similarly, organic polycations synergistically activate T903-Rhoc with di- and trivalent metals. Alanine substitution of all the acidic residues in the second extracellular loop of the T903-Rhoc receptor significantly impairs activation by metal ions and organic polycations in the presence of NPS 568 but not the synergistic activation of Ca(2+) with poly-l-Arginine. These data indicate that although the ECD has been thought to be the main determinant for Ca(2+) recognition, the 7TM core of the Ca(2+) receptor contains activating site(s) recognizing Ca(2+) and Gd(3+) as well as the allosteric modulators NPS 568 and organic polycations that may play important roles in the regulation of receptor activation.

Highlights

Kausik Ray‡ and John Northup From the Laboratory of Cellular Biology, NIDCD, National Institutes of Health, Bethesda, Maryland 20892
Unlike G protein-coupled receptors (GPCR) in the largest family with ligand binding within the seven-transmembrane (7TM) core domain, both chimeric receptor analysis and direct binding assays with purified extracellular domain (ECD) of several subtypes of mGluRs and GABAB receptor have suggested that the fusion of a periplasmic binding protein (the ECD) of these family 3 receptors is responsible for ligand recognition (6 – 8)
Signaling by the 7TM Domain of the human Ca2ϩ receptor (hCaR)—Like many family 3 GPCRs including mGluR1, mGluR2, and GABAB receptor, agonist binding seems to occur primarily within the large ECD of the hCaR rather than in the pocket defined by the 7TM helices as is characteristic of most classical rhodopsin-like GPCRs [5]

Summary

Introduction

Kausik Ray‡ and John Northup From the Laboratory of Cellular Biology, NIDCD, National Institutes of Health, Bethesda, Maryland 20892. Alanine substitution of all the acidic residues in the second extracellular loop of the T903-Rhoc receptor significantly impairs activation by metal ions and organic polycations in the presence of NPS 568 but not the synergistic activation of Ca2؉ with poly-L-Arginine These data indicate that the ECD has been thought to be the main determinant for Ca2؉ recognition, the 7TM core of the Ca2؉ receptor contains activating site(s) recognizing Ca2؉ and Gd3؉ as well as the allosteric modulators NPS 568 and organic polycations that may play important roles in the regulation of receptor activation. Chimeric receptors with the hCaR ECD-linked to the mGluR1 7TM domain confer intracellular phosphoinositide (PI) hydrolysis responsiveness to extracellular Ca2ϩ to cells expressing the constructs [11, 12] These results clearly identify the ECD as containing a Ca2ϩ recognition site(s). Metabotropic glutamate receptor; hCaR, human calcium sensing receptor; Exo-loop, second extracellular loop; PI, phosphoinositide; HEK, human embryonic kidney; IP, inositol phosphates; PIPES, 1,4-piperazinediethanesulfonic acid

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