Arora K K, Sakai A, Catt K J
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 1995 Sep 29;270(39):22820-6. doi: 10.1074/jbc.270.39.22820.
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 Ser140 to the conserved Tyr, and the conserved Leu147 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 Leu147 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 Ser140 by Tyr does not affect G protein coupling but significantly increases receptor affinity and internalization rate. In contrast, replacement of a conserved aliphatic residue (Leu147) impairs both G protein coupling and agonist-induced receptor internalization.
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环（2i环）中具有高度保守的DRYXXV/IXXPL序列，该序列与G蛋白偶联有关。GnRH受体预测的2i环在DRY序列中含有丝氨酸而非酪氨酸，但保留了保守的疏水性亮氨酸残基，这是G蛋白偶联和毒蕈碱受体内化所必需的。本研究检测了将独特的Ser140突变为保守的Tyr，以及将保守的Leu147突变为Ala或Asp对激动剂结合、内化和信号转导的影响。S140Y突变体的激动剂结合亲和力增加了100%，其内化比野生型受体增加了60%。Leu147突变体的结合特性与野生型受体无异，但其内化减少了约50%。L147A和L147D突变体还显示GnRH刺激的肌醇磷酸生成显著受损。这些发现表明，用Tyr替代Ser140不影响G蛋白偶联，但显著增加受体亲和力和内化率。相反，保守脂肪族残基（Leu147）的替换损害了G蛋白偶联和激动剂诱导的受体内化。
