RANDOM MUTAGENESIS OF THE CAMP CHEMOATTRACTANT RECEPTOR, CAR1, OF DICTYOSTELIUM - EVIDENCE FOR MULTIPLE STATES OF ACTIVATION

cAMP receptor 1 (cAR1) of Dictyostelium couples to the G protein G2 to mediate activation of adenylyl and guanylyl cyclases, chemotaxis, and cell aggregation. Other cAR1-dependent events, including receptor pho sphorylation and influx of extracellular Ca2+, do not require G protei ns, To further characterize signal transduction through cAR1, we perfo rmed random mutagenesis of the third intracellular loop (24 amino acid s), since the corresponding region of other seven helix receptors has been implicated in the coupling to G proteins. Mutant receptors were e xpressed in car1(-) cells and were characterized for G protein-depende nt and -independent signal transduction. Our results demonstrate that cAR1 is remarkably tolerant to amino acid substitutions in the third i ntracellular loop. Of the 21 positions where amino acid substitutions were observed, one or more replacements were found that retained full biological function. However, certain alterations resulted in receptor s with reduced ability to bind cAMP and/or transduce signals, There we re specific signal transduction mutants that could undergo cAMP-depend ent cAR1 phosphorylation but were impaired either in coupling to G pro teins, in G protein-independent Ca2+ influx, or in both pathways. In a ddition, there were general activation mutants that failed to restore aggregation to car1(-) cells and displayed severe defects in all signa l transduction events, including the most robust response, cAMP-depend ent cAR1 phosphorylation. Certain of these mutant phenotypes were obta ined in a complementary study, where the entire region of cAR1 from th e third to the seventh transmembrane helices was randomly mutagenized. Considered together, these studies indicate that the activation cycle of cAR1 may involve a number of distinct receptor intermediates. A mo del of G protein-dependent and -independent signal transduction throug h cAR1 is discussed.