Computational analysis of the extracellular domain of the Ca²⁺-sensing receptor: an alternate model for the Ca²⁺ sensing region.

The extracellular Ca(2+) sensing receptor (CaSR) belongs to Class C G-protein-coupled receptors (GPCRs) which include receptors for amino acids, γ-aminobutyric acid and glutamate neurotransmitters. CaSR has been described as having an extended sequence containing a Ca(2+) binding pocket within an extracellular amino (N)-terminal domain, called a Venus Fly Trap (VFT) module. CaSR is thought to consist of three domains: 1) a Ca(2+-)sensory domain, 2) a region containing 7 transmembrane (TM) helices, and 3) a carboxy (C)-terminal tail. We find that SPOCTOPUS (a combination of hidden Markov models and artificial neural networks) predicts that Homo sapiens CaSR contains two additional TM helices ((190)D - G(210); (262)S-E(282)), with the second TM helix containing a pore-lining region ((265)K - I(280)). This predicts that the putative Ca(2+) sensory domain is within an extracellular loop, N-terminal to the highly conserved heptahelical bundle. This loop contains both the cysteine-rich domain ((537)V - C(598)) and a 14 residue "linker" sequence ((599)I - F(612)) thought to support signal transmission to the heptahelical bundle. Thus domain 1 may contain a 189 residue N-terminal extracellular region followed successively by TM-1, a short intracellular loop, TM-2 and a 329 residue extracellular loop; rather than the proposed 620 residue VFT module based on crystallography of the N-terminal region of mGluR1. Since the topologies of the two proteins differ, the published CaSR VFT model is questionable. CaSR also contains multiple caveolin-binding motifs and cholesterol-binding (CRAC/CARC) domains, facilitating localization to plasma membrane lipid rafts. Ion sensing may involve combination of pore-lining regions from CaSR dimers and CaSR-bound caveolins to form ion channels capable of monitoring ionized Ca(2+) levels.