Characterization of GRK2-catalyzed phosphorylation of the human substance P receptor in Sf9 membranes.
Characterization of GRK2-catalyzed phosphorylation of the human substance P receptor in Sf9 membranes.
复制标题
Sf9 膜中 GRK2 催化的人物质 P 受体磷酸化的表征。
DOI：
10.1021/bi972302s
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Kwatra,MM
中科院分区：
文献类型：
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作者：
Nishimura,K;Warabi,K;Roush,ED;Frederick,J;Schwinn,DA;Kwatra,MM
G 蛋白偶联受体激酶 (GRK) 磷酸化激动剂占据的 G 蛋白偶联受体 (GPCR)，导致 GPCR 脱敏。 GRK2 是六种已知 GRK 中研究较好的之一，它可以磷酸化多个 GPCR。在之前的一项研究中，我们记录了 GRK2 和 GRK3 磷酸化纯化和重建的大鼠 P 物质受体 (rSPR) [Kwatra 等人。 (1993)J。生物。化学268, 9161−9164]。在这里，我们详细描述了完整膜中 GRK2 催化的人 SPR (hSPR) 磷酸化。 GRK2 以激动剂依赖性方式磷酸化尿素洗涤的 Sf9 膜中的 hSPR，其化学计量为 19 ± 1 mol 磷酸盐/mol 受体，在 Gβγ 存在时略有增加（增加 1.3 倍）。动力学分析表明，受体磷酸化发生的 aKm 为 6.3 ± 0.4 nM，aVmax 为 1.8 ± 0.1 nmol/min/mg；这些动力学参数仅受 Gβγ [Km= 3.6 ± 1.0 nM 和 Vmax= 2.2 ± 0.2 nmol/min/mg] 的轻微影响。 Gβγ 对 GRK2 催化的 hSPR 磷酸化缺乏强烈的刺激作用是令人惊讶的，因为 Gβγ 有效刺激 GRK2 催化的 β2-肾上腺素受体和视紫红质的磷酸化。膜中内源性 Gβγ 的参与被排除为高水平 hSPR 磷酸化的来源，因为受体磷酸化不受抑制或增强内源性 Gβγ 释放的鸟嘌呤核苷酸的影响。本研究首次确定了完整膜中 GRK2 受体底物磷酸化的动力学。此外，我们的结果确定 hSPR 是 GRK2 的独特底物，即使在没有 Gβγ 的情况下，其磷酸化也很强。
G protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupied G protein-coupled receptors (GPCRs), resulting in GPCR desensitization. GRK2 is one of the better studied of the six known GRKs and phosphorylates several GPCRs. In a previous study, we documented that GRK2 and GRK3 phosphorylate purified and reconstituted rat substance P receptor (rSPR) [Kwatra et al. (1993)J. Biol. Chem.268, 9161−9164]. Here, we characterize in detail GRK2-catalyzed phosphorylation of human SPR (hSPR) in intact membranes. GRK2 phosphorylates hSPR in urea-washed Sf9 membranes in an agonist-dependent manner with a stoichiometry of 19 ± 1 mol of phosphate/mol of receptor, which increases slightly (1.3-fold increase) in the presence of Gβγ. Kinetic analyses indicate that receptor phosphorylation occurs with aKmof 6.3 ± 0.4 nM and aVmaxof 1.8 ± 0.1 nmol/min/mg; these kinetic parameters are only slightly affected by Gβγ [Km= 3.6 ± 1.0 nM andVmax= 2.2 ± 0.2 nmol/min/mg]. The lack of a strong stimulatory effect of Gβγ on GRK2-catalyzed phosphorylation of hSPR is surprising since Gβγ potently stimulates GRK2-catalyzed phosphorylation of β2-adrenergic receptor and rhodopsin. Involvement of Gβγ endogenously present in membranes is ruled out as a source of high levels of hSPR phosphorylation, since receptor phosphorylation was not affected by guanine nucleotides that suppress or enhance the release of endogenous Gβγ. The present study determines, for the first time, the kinetics of phosphorylation of a receptor substrate of GRK2 in intact membranes. Further, our results identify hSPR as a unique substrate of GRK2 whose phosphorylation is strong even in the absence of Gβγ.