EFFECTS OF 2ND INTRACELLULAR LOOP MUTATIONS ON SIGNAL-TRANSDUCTION AND INTERNALIZATION OF THE GONADOTROPIN-RELEASING-HORMONE RECEPTOR
EFFECTS OF 2ND INTRACELLULAR LOOP MUTATIONS ON SIGNAL-TRANSDUCTION AND INTERNALIZATION OF THE GONADOTROPIN-RELEASING-HORMONE RECEPTOR
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DOI：
10.1074/jbc.270.39.22820
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发表时间：
1995-09-29
影响因子：
4.8
通讯作者：
CATT, KJ
中科院分区：
文献类型：
--
作者：
ARORA, KK;SAKAI, A;CATT, KJ
促性腺激素释放激素（GnRH）受体属于七螺旋G蛋白偶联受体超家族，其中大多数在第二个细胞内（2 i）环中具有高度保守的DRYXXV/IXXPL序列，该序列与G蛋白偶联有关。预测的GnRH受体2 i环在DRY序列中含有丝氨酸而不是酪氨酸，但保留了保守的疏水性Leu残基，这是G蛋白偶联和毒蕈碱受体内化所需的。本研究检测了将独特的Ser（140）突变为保守的Tyr，以及将保守的Leu（147）突变为Ala或Asp对激动剂结合、内化和信号转导的影响。S140 Y突变体显示激动剂结合亲和力增加100%，并且其内化比野生型受体增加60%。Leu（147）突变体的结合特性与野生型受体的结合特性没有区别，但它们的内化减少了约50%。L147 A和L147 D突变体对GnRH刺激的磷酸肌醇合成也有明显的抑制作用。这些结果表明Tyr取代Ser（140）不影响G蛋白偶联，但显著增加受体亲和力和内化速率。相反，保守的脂肪族残基（Leu（147））的替代损害G蛋白偶联和激动剂诱导的受体内化。
The gonadotropin-releasing hormone (GnRH) receptor belongs to the superfamily of heptahelical G protein coupled receptors, most of which have a highly conserved DRYXXV/IXXPL sequence in the second intracellular (2i) loop that has been implicated in G protein coupling. The predicted 2i loop of the GnRH receptor contains serine rather than tyrosine in the DRY sequence but retains the conserved hydrophobic Leu residue, which is required for G protein coupling and internalization of muscarinic receptors. The present study examined the effects of mutating the unique Ser(140) to the conserved Tyr, and the conserved Leu(147) to Ala or Asp, on agonist binding, internalization, and signal transduction. The S140Y mutant showed a 100% increase in agonist binding affinity, and its internalization was increased by 60% above that of the wild-type receptor. The binding characteristics of the Leu(147) mutants were indistinguishable from those of the wild-type receptor, but their internalization was reduced by about 50%. The L147A and L147D mutants also showed significant impairment of GnRH-stimulated inositol phosphate production, These findings demonstrate that substitution of Ser(140) by Tyr does not affect G protein coupling but significantly increases receptor affinity and internalization rate. In contrast, replacement of a conserved aliphatic residue (Leu(147)) impairs both G protein coupling and agonist-induced receptor internalization.