Human Ca2+ Receptor Extracellular Domain

Abstract

The G protein-coupled Ca 2؉ 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. 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 ] o homeostasis: familial hypocalciuric hypercalcemia and autosomal dominant hypocalcemia, respectively (2).The CaR is a member of family 3 of the superfamily of G protein-coupled receptors (3). In addition to amino acid sequence conservation, the CaR shares with other members of family 3, such as the metabotropic glutamate receptors (mGluR), a unique structural feature, namely a large ϳ600-residue extracellular amino-terminal domain (ECD). The ECD in typical family 3 members consists of an ϳ530-residue domain, which based on limited homology to bacterial periplasmic-binding proteins has been suggested to resemble a bilobed Venus's-flytrap (VFT) (4), followed by an ϳ70-residue cysteinerich domain (5).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). The model shows a bilobed VFT with lobe I (containing the amino terminus) connected by three strands to lobe II (containing the carboxyl terminus). Four insertions in the hCaR and mGluR1 sequence that could not be aligned with leucine/isoleucine/ valine bacterial periplasmic-binding protein could not be modeled, and hence these were shown as unstructured loops (designated I-IV). Loops I-IV were all contained in lobe I in our model. We also 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).Recently, Kunishima et al. (7) determined the three-dimensional structure of residues 33-522 from the ECD of the rat mGluR1 in two free (unliganded) forms and in a glutamatebound form. All three forms appear as intermolecular disulfidelinked homodimers with each monomer consisting of a bilobed VFT. The flytrap is closed in the ligand-bound state and either open (free form I) or closed (free form II) in the unliganded state. The sequences corresponding to loops I-IV in our hCaR model are all confirmed to occur in lobe I, and for loops I, III, and IV are shown to be loops connecting regions of secondary structure in the mGluR1 crystal structure. Loop II, which was confirmed to be the site of covalent dimerization (involving the sin...

A Double Mutation in the Extracellular Ca2+-sensing Receptor's Venus Flytrap Domain That Selectively Disables l-Amino Acid Sensing

“…The mutagenic primers were designed to insert the FLAG tag between CaR amino acid residues 371 and 372. This site was selected after Bai et al (13) and lies in a loop of the VFT domain that does not contribute significantly to ligand binding or receptor activation (14). The sequences of the mutagenic primers (with the complementary sequences encoding the FLAG tag underlined) were 5Ј-GCAAAAGGACCTTTAC-CTGTGGACTACAAGGATGACGATGACAAGACCTTTCTGAGAGGT-CACGAAGAAAGTGG-3Ј and 5Ј-CCACTTTCTTCGTGACCTCTCAGA-AAGGTCTTGTCATCGTCATCCTTGTAGT CCACAGGTAAAGGTCC-TTTTGC-3Ј.…”

Molecular Basis of the Extracellular Ligands Mediated Signaling by the Calcium Sensing Receptor

“…Figure 2 shows three modeled structures of the ECD of CaSR in different conformational states based on the crystal structure of mGluRs. The ECD of CaSR possesses a featured bilobed VFT structure similar to mGluRs and bacterial periplasmic binding proteins (O'Hara et al, 1993 ; Ray et al, 1999 ; Reyes-Cruz et al, 2001 ). These modeled structures of CaSR derived from several crystal structures of mGluR1 provide molecular views of large conformational variances among different states and therefore provide a potential functional mechanism of CaSR and by extension, of other cGPCRs.…”

Modeling and Mutagenesis of the Binding Site of Calhex 231, a Novel Negative Allosteric Modulator of the Extracellular Ca2+-sensing Receptor

“…We used molecular modeling approaches, mutagenesis, and functional activity (phospholipase C) to identify for the first time residues involved in the binding pocket of a negative modulator of the CaSR. The amino-terminal domain of the CaSR is thought to contain the Ca 2ϩ -binding sites and has been submitted to extensive mutation and deletion studies, which have given insight on the mechanism of CaSR activation (41)(42)(43). However, little is known about the binding sites of positive or negative allosteric modulators of the CaSR.…”