Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases.


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.







Neil Jonathan Freedman

Professor of Medicine

Our work focuses on atherosclerosis-related signal transduction and the genetic bases of atherosclerosis and vein graft failure, both in vitro and in vivo. We investigate the regulation of receptor protein tyrosine kinases by G protein-coupled receptor kinases (GRKs), and the role of GRKs and β-arrestins in atherosclerosis; the role of tumor necrosis factor and its receptors in atherosclerosis; and the role of the dual Rho-GEF kalirin in atherosclerosis. For in vivo modeling of atherosclerosis and neointimal hyperplasia, we use mouse carotid artery bypass grafting with either veins or arteries from gene-deleted or congenic wild type mice, as well as aortic atherosclerosis studies and bone marrow transplantation. To study receptor phosphorylation, signal transduction, and intracellular trafficking, we employ primary smooth muscle cells, endothelial cells, and macrophages derived from knockout mice or treated with RNA interference.

Key Words: atherosclerosis, G protein-coupled receptor kinases, arrestins, desensitization, phosphorylation, platelet-derived growth factor receptors, receptor protein tyrosine kinases, smooth muscle cells, neointimal hyperplasia, Rho-GEF.

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