MUTATION OF INDIVIDUAL SERINE RESIDUES IN THE C-TERMINAL TAIL OF THE LUTROPIN CHORIOGONADOTROPIN RECEPTOR REVEAL DISTINCT STRUCTURAL REQUIREMENTS FOR AGONIST-INDUCED UNCOUPLING AND AGONIST-INDUCED INTERNALIZATION/

We have previously mapped the agonist-induced phosphorylation of the r at lutropin/choriogonadotropin receptor (rLHR) to a locus of four seri nes (Ser(635), Ser(639), Ser(649), and Ser(652)) located in the C-term inal tail. The removal or mutation of this locus delays the time cours e of agonist-induced uncoupling of the rLHR from its effector system w ithout affecting the overall magnitude of uncoupling, and it retards t he endocytosis of the agonist-receptor complex. We have now prepared a nd analyzed four new rLHR mutants in which each of these serines were individually mutated to alanines, The data presented show that each mu tation reduces agonist-promoted rLHR phosphorylation by 20-40%. Mutati on of Ser(635) or Ser(639) delayed the time course of agonist-induced uncoupling to about the same extent as the simultaneous mutation of al l four serines. Mutation of Ser(635) or Ser(639) also retarded agonist -induced internalization, but the magnitude of this decrease was less than that induced by the simultaneous mutation of all four serines, Mu tation of Ser(649) had no effect on agonist-induced uncoupling but ret arded agonist-induced internalization to the same extent as the simult aneous mutation of all four serines. R Mutation of Ser(652) has little or no effect on either of these two parameters. Co-transfection studi es with dominant-negative arrestins and dominant-negative dynamin reve al that, despite differences in their rates of internalization, rLHR-w ild-type, rLHR-S639A, and rLHR-S649A are internalized by an arrestin- and dynamin-dependent pathway. These data show that the structural req uirements needed for the agonist-induced uncoupling and internalizatio n of the rLHR are distinct.