RANDOM MUTAGENESIS OF THE CAMP CHEMOATTRACTANT RECEPTOR, CAR1, OF DICTYOSTELIUM - MUTANT CLASSES THAT CAUSE DISCRETE SHIFTS IN AGONIST AFFINITY AND LOCK THE RECEPTOR IN A NOVEL ACTIVATIONAL INTERMEDIATE

The cAMP chemoattractant receptor, cAR1, of Dictyostelium transduces e xtracellular cAMP signals via G protein-dependent and G protein-indepe ndent mechanisms. While site directed mutagenesis studies of G protein -coupled receptors have provided a host of information regarding the d omains essential for various functions, many mechanistic and structura l questions remain to be resolved. We therefore carried out polymerase chain reaction-mediated random mutagenesis over a large part of the c AR1 sequence (from TMIII through the proximal part of the cytoplasmic tail). We devised a rapid screen for loss-of-function mutations based on the essential role of cAR1 in the developmental program of Dictyost elium. Although there were an average of two amino acid substitutions per receptor, similar to 90% of the mutants were able to substitute fo r wild-type cAR1 when expressed in receptor null cells. About 2% were loss-of-function mutants that expressed wild-type levels of receptor p rotein. We used biochemical screens to select about 100 of these mutan ts and chose eight representative mutants for extensive characterizati on. These fell into distinct classes. One class had a conditional defe ct in cAMP binding that was reversed by high salt. Another large class had decreased affinity under all conditions. Curiously, the decreases were clustered into three discrete intervals. One of the most interes ting class of mutants lost all capacity for signal transduction but wa s phosphorylated in response to agonist binding. This latter finding s uggests that there are at least two activated states of cAR1 that can be recognized by different downstream effecters.