9728179 Sterne-Marr G protein coupled receptors are membrane bound proteins which convert light and hormonal signals into intracellular messages. Uncoupling of the receptor from its immediate downstream signal transducer, the G protein, occurs rapidly following exposure to a stimulus and results in cessation of response to the hormone (desensitization). Receptor phosphorylation by the (adrenergic receptor kinase ( ARK) is hormone-dependent and promotes G protein/receptor uncoupling. Prior to hormone treatment ARK exists in an inactive state. Upon activation of the receptor by hormone, the catalytic activity of the kinase is stimulated and the receptor is phosphorylated, resulting in desensitization. Little is known about the structure of ARK, the molecular interactions which maintain the pre-active kinase in its basal state, and the conformation changes which convert the pre-active kinase into an active enzyme. The goals of this project are to understand how interactions within the ARK protein maintain this molecule in an inactive form and how interactions between ARK and a hormone stimulated receptor induce ARK activation. The techniques of mass spectrometry, chemical cross-linking and random targeted mutagenesis will be used to identify the amino acids in ARK which keep this protein in the pre-active form. Fusion proteins, site-directed mutagenesis, enzyme assays and ARK /receptor binding assays will be used to identify regions that interact with the receptors. Since ARK is a member of a family of G protein coupled receptor kinases which regulate G protein-coupled receptors, it provides a good model to study the structure and activation of this class of proteins. The interaction of a hormone or neurotransmitter with a specific receptor on the surface of a target cell initiates a chemical signaling event. A molecule known st a G protein, because it binds GTP, interacts with the receptor and downstream signaling molecules and a specific response occurs. To terminate the signal a specific protein, the adrenergic receptor kinase ( ARK) adds a phosphate group to the receptors. This research is directed at the mechanisms by which this protein becomes activated and interacts with the receptor. These studies will provide new information about activation of this class of proteins. ***
