When analysed on SDS/PAGE, the major cell surface cAMP receptor in Dicytostelium appears as a doublet of bands, designated R (MW=43000). In vivo, in the absence of extracellular cAMP, the R form predominates. Binding of cAMP to the receptor triggers its modification and, within 15 minutes, 85% migrates as the D form. The modification appears to be phosphorylation; the D form is phosphorylated at least 6-fold more intensely than the R form. The kinetics and concentration dependence of this ligand-induced receptor modification suggest that it is the mechanism that causes adaptation of the two major responses to cAMP: chemotaxis and activation of adenylate cyclase. A specific antiserum, raised against the purified receptor, will be used to screen a Lambda GTll cDNA library. Hybrid-selection, affinity purification of linked antigenic determinants, and comparison of protein and nucleotide sequence will be used to prove that positive plaques are expressing fragments of the receptor. The receptor gene will be cloned and the nucleotide sequence of genomic and cDNA compared. Basal and ligand-induced sites of receptor phosphorylation will be characterized by 2-dimensional peptide mapping of immunoprecipitated receptor phosphorylated in vivo and in vitro. The location of these sites within the primary sequence of the receptor will be determined by partial sequencing of the phosphopeptides. Preparative amounts of the critical phosphopeptides will be synthesized and used as substrates or inhibitors in in vitro assays for receptor kinase(s) and to generate anti-idiotype antisera directed against the kinase(s). The role of receptor modification in adaptation of the physiological responses will be tested by specific inhibition of in vivo receptor phosphorylation and investigation of mutants defective in this reaction. Dictyostelium cells will be transformed with altered or anti-sense receptor sequences and the effects of specific mutation or deletion of the receptor examined. These studies are designed to elucidate the role of receptors and ligand-induced receptor modification in regulating chemotaxis, activation of adenylate cyclase, and gene expression.
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