May 15, 2000-April 30, 2001
Level of Access
Guanine nucleotide-binding proteins (G proteins) are vital to a wide variety of physiological processes such as vision, hormone response, olfaction, and development. G protein-mediated signal transduction involves a complex network of receptors and second messenger pathways, and proper regulation of the network is essential for proper response of the system. A detailed analysis of the functional domains of the G protein alpha-subunit has been initiated by screening a library of random mutations in the subunit, G alpha2, of the slime mold, Dictyostelium. The function of G alpha2 function is essential for the developmental life cycle of this organism. It regulates three major response pathways in the developing cells: cyclic AMP signaling, chemotaxis, and differentiation. The library of the random G alpha2 mutants has been screened for defects in the initiation of development using a Dictyostelium cell line, MYC2, that otherwise is unable to enter the developmental stage because it lacks its own wild-type G alpha2 gene. Several G alpha2 mutations that are unable to rescue the developmental phase when transformed into MYC2 have been isolated using a series of simple phenotypic screens. The G alpha2-containing plasmids from developmental mutants were recovered and sequenced to identify the G alpha2 mutations which result in defective function. Those mutations that have not been previously identified will be examined to determine the biochemical defect. Mutants will be assayed for defects in the regulation of adenylyl cyclase, guanylyl cyclase and phospholipase C, the three major effectors activated by G alpha2. Alpha-subunit interactions with its receptor and the beta-gamma complex, as well as GTP binding and hydrolysis will also be examined. Placement of the mutations within the context of a three-dimensional model based on the alpha-subunit crystal structure of transducin will be used to assign functional roles to the mutated amino acid residues. The G alpha2 sequences that contain multiple mutations (roughly half), will be modified through site-directed mutagenesis to isolate the causative mutation.
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Gundersen, Robert E., "Identification of Functional Amino Acids in the G Protein Alpha-Subunit" (2001). University of Maine Office of Research Administration: Grant Reports. 256.