Many hormones, autacoids, neuroregulatory agents, and sensory stimuli act through cell-surface receptors that are coupled to intracellular guanine nucleotide-binding proteins (G proteins). Receptors of this family mediate diverse physiological processes in man, including regulation of blood pressure, inflammation, cardiac rhythm, synaptic response and neuronal plasticity, sensation of light and olfactants, and regulation of cellular proliferation. The mechanisms and regulation of signal transduction between receptors, G proteins and their intracellular effectors are unresolved issues of key importance from the standpoint of developing pharmacological agents targeted to specific receptor subtypes, and understanding (and perhaps controlling) the transforming activity of certain cellular oncogenes. Although the structural features of receptors and G proteins that mediate transmembrane signaling are beginning to emerge, the mechanisms by which receptors, G proteins and effectors are activated and regulated are poorly understood. The long-term goal of this research program is to establish the fundamental molecular principles by which G protein-coupled signaling systems are regulated. for these purposes a system offering unique experimental advantages will be used: the alpha-factor mating pheromone signal transduction pathway of the yeast Saccharomyces cerevisiae. Genes encoding the alpha-factor receptor and the alpha, beta and gamma subunits of its cognate G protein have been identified. Pharmacological and biochemical assays for receptor/G protein function are now well-developed. These biochemical techniques will be combined with those of classical yeast genetics, molecular genetics and yeast mating physiology to select and characterize mutant receptors and G protein subunits that alter the signal transduction process in novel and illuminating ways. Also, the potential nature of intracellular effectors will be explored. Specific objectives are to: 1. Identify structural elements of the alpha- factor receptor involved in transmembrane signaling by selecting for receptor variants that: i) no longer activate G protein; ii) constitutively activate G protein without binding agonist; and iii) have gained the ability to respond to alpha-factor antagonists. 2. Establish whether receptor mutations affect G protein interaction or activation by using: i) techniques of allele-specific suppression to obtain G protein variants that define specific protein-protein interactions; ii) kinetic, equilibrium, and competition binding experiments to monitor receptor-G protein coupling in vitro; iii) of agonist-stimulated GTPase or GDP/GTP exchange to monitor G protein activation. 3. Explore the potential intracellular second messenger system(s) that mediate response to alpha- factor by determining whether: i) the alpha-factor receptor and its cognate yeast G protein subunits expressed in Xenopus oocytes modulate effectors that regulate ion channel activity; and ii) yeast cell stimulated with alpha-factor produce putative second messengers through the hydrolysis of membrane phospholipids.
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