Extracellular cAMP plays multiple roles in Dictyostelium development, acting as a chemoattractant, a cell-cell signaling molecule, a morphogen, and an inducer of gene expression. We have recently discovered that Dictyostelium sequentially expresses multiple surface cAMP receptors (cARs) during its developmental program. The discovery of multiple cAR subtypes may explain the versatility of cAMP. The cARs resemble mammalian G-protein linked receptors such as rhodopsin, the beta-adrenergic receptor, and the mucarinic acetylcholine receptor. The cAR subtypes are strongly homologous throughout the putative seven membrane spanning domains and connecting loops but differ strikingly in the C-terminal putative cytoplasmic domains. We will complete the cloning and sequencing of three cAR subtypes (cAR1, cAR2, and cAR3), design cAR general and subtype specific probes, analyze genomic DNA blots, clone a fourth cAR subtype (cAR4), and generate specific antibodies against each subtype. We will verify that each cAR is a cAMP binding protein, determine its time course of expression, whether it undergoes ligand-induced modification, and its subcellular distribution. In order to assess the role of each cAR subtype in the developmental program of Dictyostelium, we will specifically delete or alter it. Initially, we will determine which subtypes are expressed in a cAR1 antisense cell line and then construct a specific antisense cell line for each subtype. We will use homologous recombination and create subtype """"""""null"""""""" cells which we will complement with functional genes. We will clone the cAR subtypes from related species of slime molds and compare the structures of those of Dictyostelium and will also screen a variety of animal species to determine whether they contain surface cAMP receptors. We will characterize those functions cAR1 carries out when expressed outside of its normal developmental context in growing cells. Its pharmacological specificity, kinetics of binding, site of attachment of photoaffinity labels, ligand-induced phosphorylation and sites of serine phosphorylation, and ligand-induced-down-regulation and redistribution will be explored. In order to assess the coupling of cAR1 to G-alpha-2, we will co-express the two proteins in growing cells. We are designing structure- function studies to determine which regions of the receptor participate in each function: 3'-deletions to roughly localize important sites, chimeric receptors to more precisely localize the sites, and site-directed and random point mutations to refine the models. In conjunction with these studies, we will carry out biophysical characterization of the """"""""cytoplasmic"""""""" domains of each receptor.
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