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 electropho retic mobility, and loss of ligand binding of a guanine nucleotide-bin ding regulatory protein (G protein)-coupled chemoattractant receptor f rom Dictyostelium (cAR1) depend upon a cluster of five C-terminal doma in serine residues (Caterina, M. J., Hereld, D., and Devreotes, P. N. (1995) J. Biol. Chem. 270, 4418-4423). Analysis of mutants lacking com binations of these serines revealed that either Ser(303) or Ser(304) i s required; mutants lacking both serines are defective in all of these responses. Interestingly, several mutants, in eluding those substitut ed at only Ser(299), Ser(302), or Ser(303) or at non-serine positions within the third cytoplasmic loop, displayed an unstable mobility shif t; the alteration was rapidly reversed upon cAMP removal. These mutant s 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 concentra tion and duration of agonist pretreatment. This suggests that, followi ng phosphorylation of Ser(303) or Ser(304), cAR1 undergoes a further t ransition (EC(50) approximate to 140 nM, t(1/2) approximate to 4 min) to a relatively phosphatase resistant state. We used this insight to s how that, under all conditions tested, the extent of loss of binding i s correlated with the fraction of cAR1 in the altered mobility form. W e discuss possible relationships between cAR1 phosphorylation and loss of ligand binding.