Expression, purification, and characterization of the G protein-coupled receptor kinase GRK6.
Expression, purification, and characterization of the G protein-coupled receptor kinase GRK6.
复制标题
DOI：
--
复制
发表时间：
1994-09
期刊：
影响因子：
--
通讯作者：
R. Loudon;J. Benovic
中科院分区：
文献类型：
--
作者：
R. Loudon;J. Benovic
G蛋白偶联受体激酶（GRKs），如视紫红质激酶和β -肾上腺素能受体激酶（β ARK），参与介导激动剂特异性磷酸化和G蛋白偶联受体的脱敏。GRK6是最近发现的GRK家族成员，与GRK5（70.1%的氨基酸同源性）和IT11（68.5%）的同源性高于β ARK（37.4%）和rhodopsin kinase（47.1%）。为了进一步表征GRK6，我们在Sf9细胞中过表达GRK6，并通过SP-Sepharose和肝素- sepharose柱的序列层析纯化GRK6。GRK6与GRK5具有许多体外特征，包括肝素和硫酸葡聚糖的强抑制作用（IC50值分别约为15 nM和7 nM），多阳离子的过度刺激，以及对非酸性肽磷酸化的偏好。视紫红质和β 2-肾上腺素能受体和m2毒蕈碱胆碱能受体作为GRK6的刺激依赖性底物，但其化学计量量明显低于GRK5和β ARK。此外，GRK6不经历显著的自磷酸化，即使它含有与GRK5和视紫红质激酶中自磷酸化的残基相同的残基。这些数据扩展了我们对不断增长的受体特异性激酶家族的认识，并表明GRK6具有不同于β ARK、视紫红质激酶和GRK5的底物特异性。
G protein-coupled receptor kinases (GRKs), such as rhodopsin kinase and beta-adrenergic receptor kinase (beta ARK), are involved in mediating agonist-specific phosphorylation and desensitization of G protein-coupled receptors. GRK6 is the most recently identified member of the GRK family and displays higher homology with GRK5 (70.1% amino acid identity) and IT11 (68.5%) compared to beta ARK (37.4%) and rhodopsin kinase (47.1%). To further characterize GRK6, it has been overexpressed in Sf9 cells and purified to homogeneity by sequential chromatography on SP-Sepharose and heparin-Sepharose columns. GRK6 shares a number of in vitro characteristics with GRK5, including potent inhibition by heparin and dextran sulfate (IC50 values of approximately 15 and approximately 7 nM, respectively), hyperstimulation by polycations, and preference for phosphorylation of non-acidic peptides. Rhodopsin and the beta 2-adrenergic and m2 muscarinic cholinergic receptors serve as stimulus-dependent substrates for GRK6, but with stoichiometries significantly lower than achieved by GRK5 and beta ARK. Additionally, GRK6 does not undergo significant autophosphorylation even though it contains residues identical to those that are autophosphorylated in GRK5 and rhodopsin kinase. These data extend our knowledge of a growing family of receptor-specific kinases and suggest that GRK6 has a substrate specificity distinct from beta ARK, rhodopsin kinase, and GRK5.