Disulfide bonds in the extracellular calcium-polyvalent cation-sensing receptor correlate with dimer formation and its response to divalent cations in vitro.
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.
Ward
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DT
Division of Nephrology, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Brown
E M
EM
Harris
H W
HW
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry*; D011992:Endosomes / Q000737:chemistry; D007684:Kidney Tubules / Q000737:chemistry; D008297:Male; D008970:Molecular Weight; D017830:Octoxynol / Q000494:pharmacology; D011489:Protein Denaturation; D051381:Rats; D017207:Rats, Sprague-Dawley; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface / Q000737:chemistry*; D013696:Temperature
D002413:Cations, Divalent; D004220:Disulfides; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface; C473605:extracellular calcium cation-sensing receptor, rat; D017830:Octoxynol; D003545:Cysteine; D002118:Calcium