REGULATION OF THE RHODOPSIN TRANSDUCIN INTERACTION BY A HIGHLY CONSERVED CARBOXYLIC-ACID GROUP
REGULATION OF THE RHODOPSIN TRANSDUCIN INTERACTION BY A HIGHLY CONSERVED CARBOXYLIC-ACID GROUP
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DOI：
10.1021/bi00079a020
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发表时间：
1993-07-20
期刊：
影响因子：
2.9
通讯作者：
SAKMAR, TP
中科院分区：
文献类型：
--
作者：
FAHMY, K;SAKMAR, TP
视紫红质是G蛋白偶联受体家族的成员，其具有结构和功能同源性。在大多数这些受体中，第三跨膜区段的胞质边界处的三肽序列（Glu或Asp/Arg/Tyr）是保守的。该区域参与视紫红质以及其他受体中的G蛋白活化。保守的Glu-134的作用进行了研究，结合实时荧光测定G蛋白（transducin）激活的视紫红质的位点特异性诱变。选择测定条件，在该条件下，通过视紫红质-转导素复合物形成或GTP γ S诱导的复合物解离来确定转导素活化速率。Glu-134被Gln取代以模拟羧酸基团的质子化状态。与视紫红质相比，这种突变导致复合物形成的pH依赖性延伸到碱性范围。用Asp取代Glu-134对pH依赖性具有相反但不太明显的影响，并降低了转导素活化的总体效率。第134位残基的酸度常数（pK（a））不能直接决定复合物形成的pH敏感性，表明其他氨基酸残基对包括Glu-134的可滴定结合结构域有贡献。与此相反，GTP γ S诱导的复合物解离的pH敏感性没有改变的突变，但绝对速率受到影响。这些数据表明，质子化状态的Glu-134有利于结合视紫红质转导和Glu-134是不可滴定的视紫红质-转导复合物。
Rhodopsin is a member of a family of G protein-coupled receptors which share structural and functional homologies. A tripeptide sequence (Glu or Asp/Arg/Tyr) at the cytoplasmic border of the third transmembrane segment is conserved among most of these receptors. This region is involved in G protein activation in rhodopsin as well as in other receptors. The role of the conserved Glu-134 was studied by site-specific mutagenesis of rhodopsin in combination with a real-time fluorescence assay of G protein (transducin) activation. Assay conditions were chosen under which the transducin activation rate was determined either by rhodopsin-transducin complex formation or by GTPgammaS-induced complex dissociation. Glu-134 was replaced by Gln in order to mimic the protonated state of the carboxylic acid group. This mutation caused the pH dependency of complex formation to extend to the alkaline range as compared with rhodopsin. Replacement of Glu-134 by Asp had an opposite but less pronounced effect on the pH dependency and lowered the overall efficiency of transducin activation. The acidity constant (pK(a)) of the residue at position 134 did not directly determine the pH sensitivity of complex formation, indicating that other amino acid residues contribute to a titratable binding domain that includes Glu-134. In contrast, the pH sensitivity of GTPgammaS-induced complex dissociation was not changed by the mutations, although absolute rates were affected. The data suggest that the protonated state of Glu-134 favors binding of rhodopsin to transducin and that Glu-134 is not titratable in the rhodopsin-transducin complex.