Ward D T, Brown E M, Harris H W
Division of Nephrology, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 1998 Jun 5;273(23):14476-83. 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 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.
细胞外钙/多价阳离子传感受体（CaR）与G蛋白偶联，含有高度保守的细胞外半胱氨酸残基。用CaR特异性抗体对大鼠肾内髓集合管内体的蛋白质进行免疫印迹分析，结果显示CaR的表观分子量会因蛋白质变性条件而发生改变。在非还原条件下用SDS变性时，CaR以240 - 310 kDa的假定二聚体形式迁移。这是在巯基还原剂存在下SDS变性后观察到的CaR单体预测分子量的两倍。在蔗糖密度梯度中，Triton X - 100溶解的CaR以220 kDa的复合物形式沉降，这无法用G蛋白与CaR单体的结合来解释。用二价（Ca2 +、Mg2 +）和三价（Gd3 +）金属离子CaR激动剂处理Triton可溶的CaR，但不是单价离子（Na +），在还原条件下会使CaR在免疫印迹上的电泳迁移率部分从主要的单体形式转变为这种假定的二聚体形式，其方式类似于它们对CaR激活的功能效力的等级顺序（Gd3 + >> Ca2 + > Mg2 +）。这种Ca2 +效应可被N - 乙基马来酰亚胺预处理阻断。我们得出结论，CaR中存在的二硫键介导了二聚体的形成，这些二聚体在Triton X - 100溶液中得以保留。此外，CaR暴露于Ca2 +会诱导Triton可溶的CaR复合物内形成额外的二硫键。
