Disulfide Bonds in the Extracellular Calcium-Polyvalent Cation-sensing Receptor Correlate with Dimer Formation and Its Response to Divalent Cations in Vitro

• Published in: The Journal of biological chemistry Vol. 273; no. 23; pp. 14476 - 14483
• Main Authors: Ward, Donald T., Brown, Edward M., Harris, H. William
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 05.06.1998; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Calcium - pharmacology; Cations, Divalent - pharmacology; Conserved Sequence; Cysteine - chemistry; Dimerization; Disulfides - chemistry; Endosomes - chemistry; Kidney Tubules - chemistry; Male; Molecular Weight; Octoxynol - pharmacology; Protein Denaturation; Rats; Rats, Sprague-Dawley; Receptors, Calcium-Sensing; Receptors, Cell Surface - chemistry; Temperature
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.23.14476

Extracellular calcium/polyvalent cation-sensing receptors (CaR) couple to G proteins and contain highly conserved extracellular cysteine residues. Immunoblotting of proteins from rat kidney inner medullary collecting duct endosomes with CaR-specific antibodies reveals alterations in the apparent molecular mass of CaR depending on protein denaturation conditions. When denatured by SDS under nonreducing conditions, CaR migrates as a putative dimeric species of 240–310 kDa. This is twice the predicted molecular mass of the CaR monomer observed after SDS denaturation in the presence of sulfhydryl-reducing agents. In sucrose density gradients, Triton X-100-solubilized CaR sediments as a 220-kDa complex, not explainable by binding of G proteins to CaR monomers. Treatment of Triton-soluble CaR with divalent (Ca2+, Mg2+) and trivalent (Gd3+) metal ion CaR agonists, but not monovalent ions (Na+), partially shifts the electrophoretic mobility of CaR under reducing conditions from a predominantly monomeric to this putative dimeric species on immunoblots in a manner similar to their rank order of functional potency for CaR activation (Gd3+≫ Ca2+ > Mg2+). This Ca2+effect is blocked by pretreatment withN-ethylmaleimide. We conclude that disulfide bonds present in CaRs mediate formation of dimers that are preserved in Triton X-100 solution. In addition, CaR exposure to Ca2+induces formation of additional disulfide bonds within the Triton-soluble CaR complex.