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J Biol Chem, Vol. 273, Issue 23, 14476-14483, June 5, 1998

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

Donald T. Ward, Edward M. Brown^§, and H. William Harris

From the  Division of Nephrology, Children's Hospital and the ^§ Endocrine-Hypertension Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115

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 with N-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.

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