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促甲状腺素释放激素受体螺旋 8在 G 蛋白偶联受体激酶磷酸化中的作用。

Role of helix 8 of the thyrotropin-releasing hormone receptor in phosphorylation by G protein-coupled receptor kinase.

机构信息

Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY 14642, USA.

出版信息

Mol Pharmacol. 2010 Feb;77(2):288-97. doi: 10.1124/mol.109.059733. Epub 2009 Nov 11.

Abstract

The thyrotropin-releasing hormone (TRH) receptor undergoes rapid and extensive agonist-dependent phosphorylation attributable to G protein-coupled receptor (GPCR) kinases (GRKs), particularly GRK2. Like many GPCRs, the TRH receptor is predicted to form an amphipathic helix, helix 8, between the NPXXY motif at the cytoplasmic end of the seventh transmembrane domain and palmitoylation sites at Cys335 and Cys337. Mutation of all six lysine and arginine residues between the NPXXY and residue 340 to glutamine (6Q receptor) did not prevent the receptor from stimulating inositol phosphate turnover but almost completely prevented receptor phosphorylation in response to TRH. Phosphorylation at all sites in the cytoplasmic tail was inhibited. The phosphorylation defect was not reversed by long incubation times or high TRH concentrations. As expected for a phosphorylation-defective receptor, the 6Q-TRH receptor did not recruit arrestin, undergo the typical arrestin-dependent increase in agonist affinity, or internalize well. Lys326, directly before phenylalanine in the common GPCR motif NPXXY(X)(5-6)F(R/K), was critical for phosphorylation. The 6Q-TRH receptor was not phosphorylated effectively in cells overexpressing GRK2 or in in vitro kinase assays containing purified GRK2. Phosphorylation of the 6Q receptor was partially restored by coexpression of a receptor with an intact helix 8 but without phosphorylation sites. Phosphorylation was inhibited but not completely prevented by alanine substitution for cysteine palmitoylation sites. Positively charged amino acids in the proximal tail of the beta2-adrenergic receptor were also important for GRK-dependent phosphorylation. The results indicate that positive residues in helix 8 of GPCRs are important for GRK-dependent phosphorylation.

摘要

促甲状腺素释放激素(TRH)受体经历快速和广泛的激动剂依赖性磷酸化归因于 G 蛋白偶联受体(GPCR)激酶(GRK),特别是 GRK2。像许多 GPCR 一样,TRH 受体被预测在第七跨膜域细胞质末端的 NPXXY 基序和半胱氨酸 335 和 337 处的棕榈酰化位点之间形成一个两亲性螺旋,螺旋 8。将 NPXXY 和残基 340 之间的六个赖氨酸和精氨酸残基突变为谷氨酰胺(6Q 受体)不会阻止受体刺激肌醇磷酸盐周转率,但几乎完全阻止受体对 TRH 的磷酸化。细胞质尾部的所有磷酸化位点均被抑制。磷酸化缺陷不能通过长时间孵育或高 TRH 浓度逆转。如预期的磷酸化缺陷受体,6Q-TRH 受体不能募集阻滞蛋白,经历典型的阻滞蛋白依赖性激动剂亲和力增加,或不能很好地内化。直接在 NPXXY(X)(5-6)F(R/K)常见 GPCR 基序中的苯丙氨酸之前的赖氨酸 326 对于磷酸化至关重要。在过度表达 GRK2 的细胞中或在包含纯化 GRK2 的体外激酶测定中,6Q-TRH 受体不能有效地被磷酸化。通过与具有完整螺旋 8 但没有磷酸化位点的受体共表达,6Q 受体的磷酸化部分得到恢复。通过用半胱氨酸棕榈酰化位点的丙氨酸取代,磷酸化被抑制但不是完全阻止。β2-肾上腺素能受体近端尾部的带正电荷的氨基酸对于 GRK 依赖性磷酸化也很重要。结果表明,GPCR 螺旋 8 中的正电荷残基对于 GRK 依赖性磷酸化很重要。

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