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 extracellular cAMP signals via G protein-dependent and G protein-independent mechanisms. While site-directed mutagenesis studies of G protein-coupled receptors have provided a host of information regarding the domains essential for various functions, many mechanistic and structural questions remain to be resolved. We therefore carried out polymerase chain reaction-mediated random mutagenesis over a large part of the cAR1 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 Dictyostelium. Although there were an average of two amino acid substitutions per receptor, approximately 90% of the mutants were able to substitute for wild-type cAR1 when expressed in receptor null cells. About 2% were loss-of-function mutants that expressed wild-type levels of receptor protein. We used biochemical screens to select about 100 of these mutants and chose eight representative mutants for extensive characterization. These fell into distinct classes. One class had a conditional defect 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 interesting class of mutants lost all capacity for signal transduction but was phosphorylated in response to agonist binding. This latter finding suggests that there are at least two activated states of cAR1 that can be recognized by different downstream effectors.
Kim
J Y
JY
Department of Biological Chemistry, The Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205, USA.
Caterina
M J
MJ
Milne
J L
JL
Lin
K C
KC
Borleis
J A
JA
Devreotes
P N
PN
D000818:Animals; D004023:Dictyostelium; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics*; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D007700:Kinetics; D016296:Mutagenesis; D010641:Phenotype; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011953:Receptors, Cyclic AMP / Q000737:chemistry / Q000235:genetics*
D005656:Fungal Proteins; D011953:Receptors, Cyclic AMP; C068125:cyclic AMP receptor cAR1; D019204:GTP-Binding Proteins