MONOCLONAL-ANTIBODIES REVEAL RECEPTOR SPECIFICITY G-PROTEIN-COUPLED RECEPTOR KINASES

Guanine nucleotide-binding regulatory protein (G protein)-coupled rece ptor kinases (GRKs) constitute a family of serine/threonine kinases th at play a major role in the agonist-induced phosphorylation and desens itization bf G-protein-coupled receptors. Herein we describe the gener ation of monoclonal antibodies (mAbs) that specifically react with GRK 2 and GRK3 or with GRK4, GRK5, and GRK6. They are used in several diff erent receptor systems to identify the kinases that are responsible fo r receptor phosphorylation and desensitization. The ability of these r eagents to inhibit GRK-mediated receptor phosphorylation is demonstrat ed in permeabilized 293 cells that overexpress individual GRKs and the type 1A angiotensin II receptor. We also use this approach to identif y the endogenous GRKs that are responsible for the agonist-induced pho sphorylation of epitope-tagged beta(2)-adrenergic receptors (beta(2)AR s) 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-s pecific mAbs have no effect. Consistent with the operation of a beta A R 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 senso ry neurons, which have been shown to express a GRK3-like protein, abol ishes desensitization of the alpha(2)AR-mediated calcium current inhib ition. The intracellular inhibition of endogenous GRKs by mAbs represe nts a novel approach to the study of receptor specificities among GRKs that should be widely applicable to many G-protein-coupled receptors.