Brown and colleagues (Nature 1993) have cloned a novel calcium-sensing receptor (CaR) which is a member of the G protein-coupled receptor (GPCR) superfamily, specifically subfamily 3 which includes metabotropic glutamate, GABA-B, taste, and putative pheromone receptors. The CaR is expressed in a variety of cell types including kidney, brain, thyroid C cells, and most prominently parathyroid cells, and is involved in extracellular calcium homeostasis. The CaR cDNA predicts a 7 transmembrane core typical of GPCR but with a large (approximately 600 residue) N-terminal extracellular domain (ECD). We are studying the receptor's structure and function in order to understand how calcium binding to the receptor leads to G protein activation. We have raised monoclonal antibodies to synthetic peptides corresponding to sequences in the ECD of the receptor. These antibodies have proved very useful in immunoblot and immunocytochemistry studies of the receptor. We have also succeeded in expressing and purifying the ECD,and in generating monoclonal antibodies against the purified ECD. These antibodies have interesting functional effects on the CaR, and are being evaluated for their epitopes to help define receptor structure/function. Biochemical characterization of the ECD included N-terminal sequencing to define site of signal peptide cleavage, definition of carbohydrate content, secondary structure by CD, and sites of tryptic cleavage. We found that the ECD is an intermolecular disulfide-linked dimer that accounts for the dimeric nature of the intact receptor. Mutagenesis of ECD cysteines has defined which are essential for receptor expression and has identified the cysteines (129 and 131) responsible for receptor dimerization. Disulfide mapping of the ECD using cleavage of the purified protein followed by mass spectrometry is in progress. We have also identified the glycosylation sites critical for receptor expression at the cell surface. We have characterized the functional effects of missense mutations identified in subjects with autosomal dominant hypocalcemia. Most such mutations cause increased sensitivity of the receptor to calcium. We have modeled the ECD structure based on its homology to bacterial periplasmic binding proteins known to have a bilobed, """"""""venus flytrap"""""""" structure, and are testing this model using mutagenesis and biochemical approaches .We have shown that a putative loop in the ECD comprising residues ~115-139 forms the dimer interface and is critical for receptor activation. This loop is a """"""""hotspot"""""""" for naturally occurring, activating mutations. We have also shown that a cysteine-rich region at the end of the ECD forms a separate domain that plays a critical role in linking calcium activation of the ECD venus flytrap domain to activation of the 7 transmembrane domain of the CaR. We have identified three acidic residues in the second extracellular loop of the 7 transmembrane domain that regulate receptor response to calcium, and a fourth acidic residue in the third extracellular loop critical for the action of a positive allosteric modulator of the receptor. Future studies are directed at elucidating in detail the mechanism of receptor activation by calcium.

Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2002
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Indirect Cost
Name
Deafness & Other Communication Disorders
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United States
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Hu, Jianxin; McLarnon, Stuart J; Mora, Stefano et al. (2005) A region in the seven-transmembrane domain of the human Ca2+ receptor critical for response to Ca2+. J Biol Chem 280:5113-20
Hu, Jianxin; Mora, Stefano; Weber, Giovanna et al. (2004) Autosomal dominant hypocalcemia in monozygotic twins caused by a de novo germline mutation near the amino-terminus of the human calcium receptor. J Bone Miner Res 19:578-86
Spiegel, Allen M; Weinstein, Lee S (2004) Inherited diseases involving g proteins and g protein-coupled receptors. Annu Rev Med 55:27-39
Hu, Jianxin; Spiegel, Allen M (2003) Naturally occurring mutations of the extracellular Ca2+-sensing receptor: implications for its structure and function. Trends Endocrinol Metab 14:282-8
Spiegel, Allen (2003) Cell signaling. beta-arrestin--not just for G protein-coupled receptors. Science 301:1338-9
Hu, Jianxin; Mora, Stefano; Colussi, Giacomo et al. (2002) Autosomal dominant hypocalcemia caused by a novel mutation in the loop 2 region of the human calcium receptor extracellular domain. J Bone Miner Res 17:1461-9
Hu, Jianxin; Reyes-Cruz, Guadalupe; Chen, Wangzhong et al. (2002) Identification of acidic residues in the extracellular loops of the seven-transmembrane domain of the human Ca2+ receptor critical for response to Ca2+ and a positive allosteric modulator. J Biol Chem 277:46622-31
Reyes-Cruz, G; Hu, J; Goldsmith, P K et al. (2001) Human Ca(2+) receptor extracellular domain. Analysis of function of lobe I loop deletion mutants. J Biol Chem 276:32145-51
Hu, J; Reyes-Cruz, G; Goldsmith, P K et al. (2001) The Venus's-flytrap and cysteine-rich domains of the human Ca2+ receptor are not linked by disulfide bonds. J Biol Chem 276:6901-4
Hauache, O M; Hu, J; Ray, K et al. (2000) Functional interactions between the extracellular domain and the seven-transmembrane domain in Ca2+ receptor activation. Endocrine 13:63-70

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