G-protein coupled receptors control a immense variety of physiological responses and are potential targets for pharmacological intervention in numerous diseases. There are hundreds of receptors that mediate responses to hormones and neurotransmitters as well as to diverse stimuli such as light, odorants, and chemoattractants. Yet all of these receptors display a common topological structure, they may all transduce signals by the same basic processes, and many may be regulated by similar intracellular pathways. Understanding the mechanisms of activation of these important molecular switches and discovery of associated proteins that control their activities are outstanding questions for future biomedical research. A combination of genetic, biophysical, and cell biological approaches, applied to the cAMP chemoattractant receptors (cARs) in D. discoideum, are intended to elucidate the function and regulation of G-protein coupled receptors in general. Constitutively active and hypersensitive receptors will be isolated by random mutagenesis and phenotypic screening. Purified, recombinant cAR1 displays a robust agonist-induced decrease in intrinsic tryptophan fluorescence, likely indicating a global conformational change. Studies of the kinetics of cAMP binding, of the agonist-induced shape changes, and of the interactions of the purified receptors with G-proteins are designed to investigate activation. Substitution of selected tryptophans and attachment of fluorescent probes to specific cysteine and lysine residues will be used to explore local domains involved in the agonist-induced movements. To delineate the stages of receptor excitation, these studies of purified proteins will include a panel of receptors bearing point mutations that have been shown in vivo to decrease affinity, prevent activation, cause constitutive activation, or stabilize a high-affinity intermediate state. A long term goal is to determine the structure of the receptors and receptor/G-protein complexes in the presence and absence of agonists in two- and three-dimensional crystals. cAR1-GFP is uniformly distributed along the membrane of single living cells. The lateral mobility of receptors at the fronts and backs of chemotactically oriented cells and of differently phosphorylated receptors will be determined. Agonist-induced phosphorylation of cAR1 decreases its affinity but, contrary to dogma, phosphorylation is not required for response termination or chemotaxis. To discover novel pathways involved in desensitization, screens of gene-tagged cell lines for cells that continually respond to persistent stimulation are planned. One gene, designated, cAMPS-resistance A, has been isolated and the biochemical mechanisms by which cells lacking this gene circumvent desensitization are being investigated.
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