Rhodopsin phosphorylation by transiently expressed human beta ARK1: a new method for drug development

Receptor phosphorylation is a key step in the process of rapid desensitization of the beta-adrenergic and other related G-coupled receptors. A specific kinase (called beta-adrenergic receptor kinase, beta ARK) has been identified, which phosphorylates the agonist-occupied form of these receptors. We have cloned the cDNA for human beta ARK1. The full-length cDNA was inserted in an expression vector (pBJI neo) and used for the transfection of eukaryotic cells (COS7). The kinase activity of the cytosolic fraction of COS7 cells was assayed 72 hours after beta ARK1 transfection. A 40-70 fold increase in cytosolic beta ARK1 activity was observed. To validate this approach we demonstrated a different degree of kinase inhibition by various types of heparin. Our system, based on transient gene expression and in vitro phosphorylation of rhodopsin, represents a new method to screen for pharmacological agents acting on this kinase.