Members of a family of guanine nucleotide-binding regulatory proteins (G proteins) are responsible for transmission of information from many membrane-bound receptors for hormones, neurotransmitters, autacoids, and physical stimuli to their intracellular effectors. Although there are several such pathways, the best studied are those for dual (stimulatory and inhibitory) regulation of adenylyl cyclase and for light-stimulated hydrolysis of cyclic GMP in retinal rods and cones. The broad gaol of the research proposed in this application is to elucidate molecular mechanisms of transmembrane signaling in pathways that include such G proteins as crucial regulatory elements.
Specific aims can be organized with regard to the G protein or effector system to be investigated. Gs is the G protein responsible for stimulation of adenylyl cyclase activity and activation of dihydropyridine-sensitive Ca2+ channels. The alpha subunit of Gs has been synthesized in E. coli and characterized. Large amounts of recombinant Gs alpha have been purified to homogeneity for the purpose of collaborative attempts to crystallize the protein and determine its three dimensional structure. The nature of covalent modifications of Gs alpha thought to be necessary for high-affinity interactions between the G protein and its effectors will also explored. The Gi/Go family of G proteins will be studied as homogeneous products after expression in E. coli. Their basic biochemical properties (e.g., kinetics of guanine nucleotide binding and hydrolysis) will be defined, as will the specificity of their interactions with receptors (e.g., alpha and beta adrenergic) and effectors (e.g., adenylyl cyclase, K+ channels, Ca2+ channels). The specificity and mechanisms of interactions of these proteins with receptors and effectors will also be studied by expression of various mutant Gi alpha and Go alpha subunits in appropriate cultured cell lines. The significance of amino-terminal myristoylation of these alpha subunits will also be investigated. Gz is a newly appreciated G protein with unique structural features. Its biological role is unknown. Gz will be purified from bovine brain and characterized; Gz alpha will also be expressed in E. coli. Biochemical and molecular biological approaches will be taken to elucidate its functions. Newly clone cDNAs that encode individual forms of adenylyl cyclase will be expressed in appropriate cell systems, and the properties of the enzymes that they encode will be determined. Antibodies will be raised to individual forms of the protein. The functional domains of this complex membrane-bound enzyme will be studied. The significance of the """"""""channel- like"""""""" topology of adenylyl cyclase will be investigated.
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