As evidenced by the recent cloning of over 140 structurally related hormone and neurotransmitter receptors, signal transduction via seven transmembrane helix proteins is an extremely widespread phenomenon in higher mammals. When excited, these receptors activate heterotrimeric G- proteins, catalyzing exchange of GTP for GDP and dissociation of the alpha- from the (beta gamma-subunits. There are a wide variety of alpha- subunits and beta gamma-subunits which, in turn, can activate a variety of effectors. Molecular genetic studies have contributed to our understanding of the structure and function of the alpha-subunits. Mutations or modification of alpha-subunits are associated with disorders such as cholera, pituitary and thyroid tumors, pseudohypothyroidism, and McKune-Albright syndrome. The G-protein linked signal transduction strategy appeared early in the evolution of eucaryotic cells. These pathways play an fundamental role in chemotaxis, cell-cell signaling, and ligand-induced gene expression in Dictyostelium. In the course of its developmental program, this simple metazoan expresses a family of four cell surface cAMP receptors (cAR1-cAR4), eight G-protein alpha-subunits (Galpha 1-Galpha 8), a beta- subunit (Gbeta), an adenylyl cyclase (ACA), and a phospholipase C-(PLC). Targeted gene disruptions have shown that cAR1, Galpha2-, and ACA are essential for development. This system will be used in a molecular genetic study of 0-protein linked signal transduction pathways. First, complementation of galpha2- (frigid A) cells and suppression of car1- and aca- cells will provide a """"""""read out"""""""" or visual screen for site-directed and random mutants of Galpha2. The mutants will be used to assess the role of Galpha2 phosphorylation and delineate the structural domains required for interaction with surface receptors and effectors. Second, a cytosolic regulator of adenylyl cyclase (CRAC), required for GTPgammaS activation of ACA, will be cloned and its mechanism of action investigated. Third, the role of the beta- and gamma-subunits will be investigated. Additional alpha-subunits will be identified and the extent of redundancy among the multiple alpha-subunits will be determined. Finally, the essential role of G-protein linked signal transduction pathways in development will be exploited to screen for novel components of these pathways by suppressing synag and frigid mutants and screening gene tagged cell lines for new synag and frigid mutants.
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