Functional importance of transmembrane helix 6 Trp279 and exoloop 3 Val299 of rat gonadotropin-releasing hormone receptor
Functional importance of transmembrane helix 6 Trp279 and exoloop 3 Val299 of rat gonadotropin-releasing hormone receptor
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DOI：
10.1124/mol.57.3.625
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发表时间：
2000-03-01
影响因子：
3.6
通讯作者：
Counis, R
中科院分区：
文献类型：
--
作者：
Chauvin, S;Bérault, A;Counis, R
以前的研究已经确定，促性腺激素释放激素（GnRH）与其受体（GnRHR）的相互作用需要配体的N-和C-末端区域部分进入跨膜核心。通过诱变和在中国仓鼠卵巢-K1细胞中表达，研究了大鼠GnRHR跨膜螺旋6中保守芳香残基Trp（279）和exoloop 3中保守芳香残基瓦尔（299）的功能意义。与野生型相比，取代色氨酸（279）与丝氨酸或精氨酸分别导致显着减少或完全废除，配体结合，并在这两种情况下，废除GnRH诱导的磷酸肌醇生产。当瓦尔（299）简单地被Ala取代时，观察到完全不存在功能性。应当提及的是，观察到所有突变和野生型受体蛋白的表达。有趣的是，双突变体[Trp（279）Arg/瓦尔（299）Ala] GnRH HR使B-max恢复至野生型（504 +/- 43 vs. 541 +/- 41 fmol/mg蛋白），但亲和力降低（4.95 +/- 1.05 vs. 0.94 +/- 0.35 nM），GnRH未能诱导肌醇磷酸。未观察到突变对受体内化的影响。建立了GnRH与大鼠GnRHR结合的三维模型，预测Trp（279）以20埃深埋于受体的跨膜核心，与GnRH的Trp 3直接接触。相反，瓦尔（299）位于跨膜螺旋7的细胞外末端不能精确限定的区域。尽管模型不能提供任何关于观察到的两个远端残基之间的相互作用的线索，但这些数据总体上揭示了GnRHR Trp（279）和瓦尔（299）的功能重要性，并表明与GnRH Trp（3）相互作用的Trp（279）代表了结合口袋的底部。
Previous studies have established that the interaction of gonadotropin-releasing hormone (GnRH) with its receptor (GnRHR) would require partial entry of the N- and C-terminal regions of ligand into the transmembrane core. The functional significance of the conserved aromatic residue Trp(279) present in the transmembrane helix 6, and Val(299) located in exoloop 3 of the rat GnRHR was investigated by mutagenesis followed by expression in Chinese hamster ovary-K1 cells. Compared with wildtype, substitution of Trp(279) with Ser or Arg resulted in a marked reduction or total abolition, respectively, of ligand binding and, in both cases, abrogation of GnRH-induced inositol phosphate production. A total absence of functionality was observed when Val(299) was simply replaced with Ala. Mention should be made that an expression of all mutated and wild-type receptor proteins was observed. Interestingly, the double mutant [Trp(279)Arg/Val(299)Ala]GnRHR restored B-max to wild type (504 +/- 43 versus 541 +/- 41 fmol/mg protein), but with a diminished affinity (4.95 +/- 1.05 versus 0.94 +/- 0.35 nM), and GnRH failed to induce inositol phosphate. No influence of the mutations was seen on internalization of the receptor. The three-dimensional model of GnRH binding to the rat GnRHR was built predicting that Trp(279) is buried at 20 Angstrom in the transmembrane core of the receptor, directly in contact with Trp 3 of GnRH. In contrast, Val(299) is located in a region that cannot be precisely defined at the extracellular end of transmembrane helix 7. Although models cannot provide any clue concerning the observed interactivity between the two distal residues, altogether these data reveal the functional importance of both GnRHR Trp(279) and Val(299) and suggest that Trp(279), interacting with GnRH Trp(3), represents the bottom of the binding pocket.