Hormones, neurotransmitters, chemoattractants, odorants, growth factors, light and other stimuli trigger dramatic responses in target cells. Interaction of surface receptors with G-proteins, a homologous family of guanine nucleotide binding proteins, is emerging as a major transmembrane signaling mechanism. Recent observations demonstrate that G protein-linked signaling systems are present in microorganisms such as Dictyostelium. Dictyostelium is uniquely suited for studies of chemotaxis in eucaryotic cells and the role of signal transduction in development. Sequence and mRNA analysis of recently isolated cDNAs homologous to alpha- and beta-subunits of mammalian G-proteins will be extended. Screening strategies will be modified to isolate cDNAs for additional classes of alpha- and beta-subunits, as well as gamma-subunits. The genes for the G-protein subunits will be cloned. Antisera specific for each G-protein subunit will be generated with peptides and fusion proteins and used to identify the gene products and study their developmental regulation. GRP, a wild-type protein which restores guanine nucleotide regulation of adenylate cyclase to a mutant, snag 7 will be purified. Antisera to GRP will be prepared and its cDNA cloned. Complementation analysis of additional synag mutants will be carried out to determine the minimum number of genes involved in guanine nucleotide regulation of adenylate cyclase. Northern and Western blotting and gene sequencing will be applied to determine whether mutants represent defects in cloned G-protein subunits. Transformation will be used to complement mutant phenotypes and construct mutants defective in individual G-protein subunits. Strategies will include antisense expression and homologous recombination. Biochemical analysis of the mutants will address questions concerning the functions of signal transduction genes. Which gene products are required for chemotaxis, cell-cell communication, gene expression, pattern formation, and morphogenesis? What is the purpose of the variety of alpha, beta-, and gamma-subunits of G- proteins? What are the functional domains within the G-protein subunits? Does shuttling between cytosolic and membrane compartments occur during signal transduction? The unity of the signal transduction pathways now apparent throughout phylogeny suggests that the forthcoming answers will apply generally.
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