Localization of ligand-induced phosphorylation sites to serine clusters in the C-terminal domain of the Dictyostelium cAMP receptor, cAR1.
When Dictyostelium cells are stimulated with cyclic adenosine 3',5'-monophosphate (cAMP), the major surface cAMP receptor expressed in early development, cAR1, undergoes a rapid phosphorylation and parallel decrease in electrophoretic mobility which may serve to regulate the activity of this G protein-coupled receptor. Biochemical analyses indicate the electrophoretic mobility shift is caused by phosphorylation of serine residues within the C-terminal cytoplasmic domain. The 18 serines of this domain are grouped in four clusters, designated 1 to 4 (in N- to C-terminal order). Two approaches were taken to determine the distribution of phosphorylation sites among the serine clusters. First, a proteolytic analysis of the C-terminal domain was performed. Second, mutants lacking various combinations of the serine clusters were created by site-directed mutagenesis and their abilities to undergo ligand-induced modification were determined. Both approaches yielded corroborative results consistent with the following model: the stimulus induces the addition of approximately two phosphates to cluster 1 and one to cluster 2; basal phosphorylation occurs predominantly in cluster 3 and to a lesser extent in cluster 2; and cluster 4 is not phosphorylated. The phosphorylation-deficient receptor mutants should be useful for establishing the role of ligand-induced phosphorylation of cAR1 in chemotaxis, cell-cell signaling, and gene expression.
Hereld
D
D
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Vaughan
R
R
Kim
J Y
JY
Borleis
J
J
Devreotes
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology*; D004023:Dictyostelium / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D017433:Protein Structure, Secondary; D011953:Receptors, Cyclic AMP / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D015183:Restriction Mapping; D012694:Serine / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D011953:Receptors, Cyclic AMP; C068125:cyclic AMP receptor cAR1; D010768:Phosphoserine; D012694:Serine; D000242:Cyclic AMP