Random chimeragenesis of G-protein-coupled receptors. Mapping the affinity of the cAMP chemoattractant receptors in Dictyostelium.
G-protein-coupled receptors mediate a wide variety of responses to extracellular stimuli in eucaryotic cells. Binding of the ligand to these receptors is thought to involve contacts within a pocket from the sequences of these genes. A family of four surface cAMP receptors that mediate responses to secreted cAMP coordinates the developmental program of Dictyostelium. A large difference in affinity for cAMP exists between cAR1 (25 and 230 nM) and cAR2 (> 5 microns). To understand the basis for this affinity difference, we generated an extensive series of cAR1/cAR2 and cAR2/cAR1 chimeras using a technique designated "random chimeragenesis." When a linearized plasmid was transformed into Escherichia coli, tandemly positioned cAR1 and cAR2 genes crossed over at homologous regions. The cAMP binding properties and EC50 values for agonist-induced phosphorylation of each of the chimeras were characterized in order to map the domains that determine the affinity. These studies implicated a domain in the second extracellular loop in which only 5 residues differ between the two receptors as the major determinant of affinity. A secondary domain including residues 110-147 (11 residue differences) was identified as a minor determinant of affinity.
Department of Biological Chemistry, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205.
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D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism*; D004023:Dictyostelium; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000378:metabolism*; D011953:Receptors, Cyclic AMP / Q000235:genetics / Q000378:metabolism*; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism; D018000:Receptors, Peptide / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D011953:Receptors, Cyclic AMP; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D019204:GTP-Binding Proteins