TRANSMEMBRANE SIGNALING VIA RECEPTORS AND G-PROTEINS

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Strategies are proposed for the elucidation of mechanisms by which a family of membrane-associated proteins, G-proteins, mediate regulation of intracellular processes by extracellular hormones. This family of proteins includes Gs and Gi, the regulatory proteins that mediate stimulation and inhibition of adenylate cyclase, and Gt (transducin), a regulatory protein of the visual system. A fourth protein, Go, is found in high quantity in brain tissue and interacts with muscarinic receptors; the function of Go is unknown. The muscarinic receptor has been chosen as the receptor component for reconstitution studies. Thus, all of the components will be derived from bovine CNS. Purification and characterization of the G-proteins will be continuous goals. This will include refinement of current procedures and development of new methods for purification of individual subunits. Characterization includes attempts to crystallize the Alpha subunit of Go, studies of the solubility of the G-proteins in the absence of detergent, and a variety of activity measurements. These measurements will focus on the Go protein and include assessment of the role of nucleotide binding and GTPase activities in activation of the proteins. The association of the G-proteins with phospholipid vesicles will attempt to assess the role of the membrane in the action of G-proteins. The reversible association of Alphao and Alphai with vesicles that contain BetaGamma will be exploited to study the role of subunit dissociation in activation. The function of Go will be explored by attempts to use purified Go to regulate PI metabolism and ion fluxes, and as a probe to identify potential targets of regulation. Specific antigodies to Go will be used to obtain the cellular and intracellular location of this protein in neural tissue. Methods for efficient reconstitution of receptors and G-proteins in phospholipid vesicles will be an initial priority. The specificity of interaction of the receptor with different G-proteins and the requirement for various subunits will be studied. Purification of the muscarinic receptor will utilize published techniques as well as new techniques that propose to use a biotinylated-antagonist or the G-proteins as affinity probes for the receptor. The long-term goal is to reconstitute purified components (receptors, G-proteins, and regulated enzymes) in phospholipid vesicles. In this way, the mechanisms of this common signaling system can be studied from hormone to effect in a well-defined milieu.