Agonist-induced loss of ligand binding is correlated with phosphorylation of cAR1, a G protein-coupled chemoattractant receptor from Dictyostelium.
The parallel agonist-induced phosphorylation, alteration in electrophoretic mobility, and loss of ligand binding of a guanine nucleotide-binding regulatory protein (G protein)-coupled chemoattractant receptor from Dictyostelium (cAR1) depend upon a cluster of five C-terminal domain serine residues (Caterina, M. J., Hereld, D., and Devreotes, P.N. (1995) J. Biol. Chem. 270, 4418-4423). Analysis of mutants lacking combinations of these serines revealed that either Ser303 or Ser304 is required; mutants lacking both serines are defective in all of these responses. Interestingly, several mutants, including those substituted at only Ser299, Ser302, or Ser303 or at non-serine positions within the third cytoplasmic loop, displayed an unstable mobility shift; the alteration was rapidly reversed upon cAMP removal. These mutants also exhibited subnormal extents of loss of ligand binding, which is assessed after removal of the ligand. For the wild-type receptor, we found that the stability of phosphorylation depends upon the concentration and duration of agonist pretreatment. This suggests that, following phosphorylation of Ser303 or Ser304, cAR1 undergoes a further transition (EC50 approximately 140 nM, t 1/2 approximately 4 min) to a relatively phosphatase-resistant state. We used this insight to show that, under all conditions tested, the extent of loss of binding is correlated with the fraction of cAR1 in the altered mobility form. We discuss possible relationships between cAR1 phosphorylation and loss of ligand binding.
Caterina
M J
MJ
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Devreotes
P N
PN
Borleis
J
J
Hereld
D
D
D000818:Animals; D001483:Base Sequence; D000242:Cyclic AMP / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004023:Dictyostelium / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense; D011953:Receptors, Cyclic AMP / Q000819:agonists* / Q000235:genetics / Q000378:metabolism
D005656:Fungal Proteins; D008024:Ligands; D016376:Oligonucleotides, Antisense; D011953:Receptors, Cyclic AMP; C068125:cyclic AMP receptor cAR1; D000242:Cyclic AMP; D019204:GTP-Binding Proteins