Arora K K, Cheng Z, Catt K J
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Mol Endocrinol. 1997 Aug;11(9):1203-12. doi: 10.1210/mend.11.9.9968.
The GnRH receptor is an unusual member of the G protein-coupled receptor (GPCR) superfamily with several unique features. One of these is a variant of the conserved DRY motif that is located at the junction of the third transmembrane domain and the second intracellular (2i) loop of most GPCRs. In the GnRH receptor, the Tyr residue of the conserved triplet is replaced by Ser, giving a DRS sequence. The aspartate and arginine residues of the triplet are highly conserved in almost all GPCRs. The functional importance of these residues was evaluated in wild type and mutant GnRH receptors expressed in COS-7 cells. Mutants in which Asp138 was replaced by Asn or Glu were poorly expressed, but showed significantly increased internalization and exhibited augmented inositol phosphate generation to maximal agonist stimulation compared with the wild type receptor. In contrast, receptors in which Arg139 was substituted with Gln, Ala, or Ser showed reduced internalization, and the GnRH-induced inositol phosphate response for the Arg139Gln mutant was significantly impaired in proportion to its low expression level. Replacing Ser140 with Ala affected neither internalization nor signal transduction. The role of the polar amino acids at the C terminus of the 2i loop was evaluated in two additional mutants (Ser151Ala, Ser153Ala, and Ser151Ala, Ser153Ala, Lys154Gln, Glu156Gln). Both of these mutants exhibited agonist-induced inositol phosphate responses similar to that of the wild type receptor, but showed increased receptor internalization. This mutational analysis indicates that the conserved Asp and Arg residues in the DRY/S triplet make important contributions to the structural integrity of the receptor and influence receptor expression, agonist-induced activation, and internalization.
促性腺激素释放激素（GnRH）受体是G蛋白偶联受体（GPCR）超家族中一个不同寻常的成员，具有几个独特特征。其中之一是保守的DRY基序的变体，该基序位于大多数GPCR的第三个跨膜结构域与第二个细胞内（2i）环的交界处。在GnRH受体中，保守三联体中的酪氨酸残基被丝氨酸取代，形成DRS序列。该三联体中的天冬氨酸和精氨酸残基在几乎所有GPCR中都高度保守。在COS-7细胞中表达的野生型和突变型GnRH受体中评估了这些残基的功能重要性。天冬氨酸138被天冬酰胺或谷氨酸取代的突变体表达不佳，但与野生型受体相比，其内化显著增加，并且对最大激动剂刺激显示出增强的肌醇磷酸生成。相反，精氨酸139被谷氨酰胺、丙氨酸或丝氨酸取代的受体显示内化减少，并且精氨酸139谷氨酰胺突变体的GnRH诱导的肌醇磷酸反应与其低表达水平成比例地显著受损。用丙氨酸取代丝氨酸140既不影响内化也不影响信号转导。在另外两个突变体（丝氨酸151丙氨酸、丝氨酸153丙氨酸和丝氨酸151丙氨酸、丝氨酸153丙氨酸、赖氨酸154谷氨酰胺、谷氨酸156谷氨酰胺）中评估了2i环C末端极性氨基酸的作用。这两个突变体均表现出与野生型受体相似的激动剂诱导的肌醇磷酸反应，但显示出受体内化增加。这种突变分析表明，DRY/S三联体中保守的天冬氨酸和精氨酸残基对受体的结构完整性做出重要贡献，并影响受体表达、激动剂诱导的激活和内化。
