The long-range objective of our research continues to be the precise biochemical elucidation of the structural and functional components of alpha 2-adrenergic receptors that are coupled with the catecholamine- dependent cyclic GMP signal pathway. In this pursuit we have made three novel observations, which are the foundation of this proposal. First, an alpha 2-adrenergic receptor subtype, termed alpha 2GC-adrenergic receptor, has been purified and characterized and a unique brain receptor subtype has been cloned, sequenced and expressed in a mammalian cell line (COS-2A). Second, alpha-2GC receptor subtype mediates catecholamine-dependent cyclic GMP formation. The mediation occurs via a 41-kD ADP-ribosylated pertussis toxin-sensitive G-protein. Third, the hormonally dependent transmembrane migration of the cyclic GMP signal appears to be down-regulated by protein kinase C. Based on these observations, we now propose to determine the structural basis of the functional coupling of this receptor subtype with guanylate cyclase and to determine the mechanism by which protein kinase C down-regulates the receptor-mediated cyclic GMP signal. More specifically, we will a) determine the primary structure of receptor the alpha 2GC-adrenergic receptor by completing our ongoing studies on the molecular cloning of the gene for this receptor, determine the nucleotide sequence of the cloned gene, and infer the amino acid sequence of the receptor; b) develop expression systems for the receptor gene to study its ligand-binding activity; c) develop polyclonal and monoclonal antibody probes against the alpha 2GC-adrenergic receptor to facilitate its bIochemical and genetic characterization in various mammalian tissues and isolated cells such as brain, kidney, heart, blood vessels, salivary glands, and uterus; d) study the mechanism by which protein kinase C down-regulates the hormonally dependent guanylate cyclase by considering: direct or indirect phosphorylation of alpha 2GC-adrenergic receptor, or of the pertussis-toxin-sensitive G-protein. To keep the project manageable, we do not propose to explore the other attractive possibility of the phosphorylation of guanylate cyclase subunit. These proposed approaches involve the preparation of specific immunological reagents, development of defined genetic systems, and precise biochemical techniques for the developed immunological probes. Future areas of receptor-mediated signal transduction research expected to evolve from this research include the potential revelation of a family of alpha 2-adrenergic receptors, their mechanisms of interaction with second-messenger molecules, and their regulation by other receptor-mediated signals. Although the proposed research is of a most fundamental nature, its biomedical ramifications in understanding the basic mechanisms of disease processes related to neurological disorders, hypertension, endocrine abnormalities, and metabolic malfunctions are clearly envisioned.
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