Abstract
The G protein-coupled Ca(2+) receptor (CaR) possesses an approximately 600-residue extracellular domain involved in ligand binding and receptor activation. Based on an alignment of the amino acid sequence of the CaR with that of bacterial periplasmic-binding proteins, the first approximately 530 residues of the extracellular domain are believed to form a domain resembling a bilobed Venus's flytrap (VFT). Four insertions in the CaR sequence that do not align with those of bacterial periplasmic-binding proteins correspond to four loops within lobe I of the VFT. We constructed a series of deletion mutants of these four loops and tested their ability to form fully processed CaR as well as their ability to be activated by Ca(2+). As many as 21 residues (365) of loop III could be deleted without impairing receptor expression or activation. Deletion of portions of either loops I (50) or IV (438) did not impair receptor expression but significantly reduced Ca(2+) activation. Deletion of the entire loop II (117) abolished receptor expression and function, but the replacement of even a single residue within this deletion mutant led to expression of a monomeric form of the receptor showing increased Ca(2+) sensitivity but reduced maximal activation. Our results reveal that certain residues within loops I and IV are dispensable in formation of the VFT domain but are critical for Ca(2+) activation of the receptor. In contrast, the residues in loop II are critical for maintaining the inactive state of the CaR. We discuss these results in light of the recently defined crystal structure of the homologous domain of the type 1 metabotropic glutamate receptor.
Highlights
From the ‡Molecular Pathophysiology Section, NIDCD, and the ¶Center for Molecular Modeling, CIT, National Institutes of Health, Bethesda, Maryland 20892
Four insertions in the human CaR (hCaR) and mGluR1 sequence that could not be aligned with leucine/isoleucine/ valine bacterial periplasmic-binding protein could not be modeled, and these were shown as unstructured loops
Functional Properties of Loop I Deletion Mutant Receptors— Loop I in the hCaR is comprised of the residues 39 – 67 (Fig. 1), which connect -sheet “b” to ␣-helix “A” in the mGluR1 crystal structure (7)
Summary
From the ‡Molecular Pathophysiology Section, NIDCD, and the ¶Center for Molecular Modeling, CIT, National Institutes of Health, Bethesda, Maryland 20892. The G protein-coupled Ca2؉ receptor (CaR) possesses an ϳ600-residue extracellular domain involved in ligand binding and receptor activation. Based on an alignment of the amino acid sequence of the CaR with that of bacterial periplasmic-binding proteins, the first ϳ530 residues of the extracellular domain are believed to form a domain resembling a bilobed Venus’s flytrap (VFT). Four insertions in the CaR sequence that do not align with those of bacterial periplasmic-binding proteins correspond to four loops within lobe I of the VFT. Based on an alignment of the human CaR (hCaR) ECD amino acid sequence with that of the mGluR1 ECD and the Escherichia coli leucine/isoleucine/valine bacterial periplasmic-binding protein, we previously created a model of residues 36 –513 of the hCaR ECD (see figure 6 in Ref. 6). We showed that the hCaR is an intermolecular disulfide-linked homodimer and identified cysteines 129 and 131, which are located in loop II as the residues involved in covalent dimerization (6)
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