Oppermann M, Diversé-Pierluissi M, Drazner M H, Dyer S L, Freedman N J, Peppel K C, Lefkowitz R J
Howard Hughes Medical Research Institute, Duke University Medical Center, Durham, NC 27710, USA.
Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7649-54. doi: 10.1073/pnas.93.15.7649.
Guanine nucleotide-binding regulatory protein (G protein)-coupled receptor kinases (GRKs) constitute a family of serine/threonine kinases that play a major role in the agonist-induced phosphorylation and desensitization of G-protein-coupled receptors. Herein we describe the generation of monoclonal antibodies (mAbs) that specifically react with GRK2 and GRK3 or with GRK4, GRK5, and GRK6. They are used in several different receptor systems to identify the kinases that are responsible for receptor phosphorylation and desensitization. The ability of these reagents to inhibit GRK- mediated receptor phosphorylation is demonstrated in permeabilized 293 cells that overexpress individual GRKs and the type 1A angiotensin II receptor. We also use this approach to identify the endogenous GRKs that are responsible for the agonist-induced phosphorylation of epitope-tagged beta2- adrenergic receptors (beta2ARs) overexpressed in rabbit ventricular myocytes that are infected with a recombinant adenovirus. In these myocytes, anti-GRK2/3 mAbs inhibit isoproterenol-induced receptor phosphorylation by 77%, while GRK4-6-specific mAbs have no effect. Consistent with the operation of a betaAR kinase-mediated mechanism, GRK2 is identified by immunoblot analysis as well as in a functional assay as the predominant GRK expressed in these cells. Microinjection of GRK2/3-specific mAbs into chicken sensory neurons, which have been shown to express a GRK3-like protein, abolishes desensitization of the alpha2AR-mediated calcium current inhibition. The intracellular inhibition of endogenous GRKs by mAbs represents a novel approach to the study of receptor specificities among GRKs that should be widely applicable to many G-protein-coupled receptors.
鸟嘌呤核苷酸结合调节蛋白（G蛋白）偶联受体激酶（GRK）构成了一个丝氨酸/苏氨酸激酶家族，在激动剂诱导的G蛋白偶联受体磷酸化和脱敏过程中起主要作用。在此，我们描述了能与GRK2和GRK3或与GRK4、GRK5和GRK6特异性反应的单克隆抗体（mAb）的产生。它们被用于几种不同的受体系统，以鉴定负责受体磷酸化和脱敏的激酶。在过表达单个GRK和1A型血管紧张素II受体的通透293细胞中，证明了这些试剂抑制GRK介导的受体磷酸化的能力。我们还使用这种方法来鉴定内源性GRK，其负责在感染重组腺病毒的兔心室肌细胞中过表达的表位标记β2-肾上腺素能受体（β2AR）的激动剂诱导的磷酸化。在这些肌细胞中，抗GRK2/3 mAb抑制异丙肾上腺素诱导的受体磷酸化达77%，而GRK4 - 6特异性mAb则无作用。与βAR激酶介导机制的运作一致，通过免疫印迹分析以及功能测定，GRK2被鉴定为这些细胞中表达的主要GRK。将GRK2/3特异性mAb显微注射到已显示表达GRK3样蛋白的鸡感觉神经元中，可消除α2AR介导的钙电流抑制的脱敏。mAb对内源性GRK的细胞内抑制代表了一种研究GRK之间受体特异性的新方法，应广泛适用于许多G蛋白偶联受体。
