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 rat lutropin/choriogonadotropin receptor (rLHR) to a locus of four serines (Ser635, Ser639, Ser649, and Ser652) located in the C-terminal tail. The removal or mutation of this locus delays the time course of agonist-induced uncoupling of the rLHR from its effector system without affecting the overall magnitude of uncoupling, and it retards the endocytosis of the agonist-receptor complex. We have now prepared and analyzed four new rLHR mutants in which each of these serines were individually mutated to alanines. The data presented show that each mutation reduces agonist-promoted rLHR phosphorylation by 20-40%. Mutation of Ser635 or Ser639 delayed the time course of agonist-induced uncoupling to about the same extent as the simultaneous mutation of all four serines. Mutation of Ser635 or Ser639 also retarded agonist-induced internalization, but the magnitude of this decrease was less than that induced by the simultaneous mutation of all four serines. Mutation of Ser649 had no effect on agonist-induced uncoupling but retarded agonist-induced internalization to the same extent as the simultaneous mutation of all four serines. Mutation of Ser652 has little or no effect on either of these two parameters. Co-transfection studies with dominant-negative arrestins and dominant-negative dynamin reveal that, despite differences in their rates of internalization, rLHR-wild-type, rLHR-S639A, and rLHR-S649A are internalized by an arrestin- and dynamin-dependent pathway. These data show that the structural requirements needed for the agonist-induced uncoupling and internalization of the rLHR are distinct.
Lazari
M F
MF
Department of Pharmacology, The University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Bertrand
J E
JE
Nakamura
K
K
Liu
X
X
Krupnick
J G
JG
Benovic
J L
JL
Ascoli
M
M
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000818:Animals; D019393:Arrestin / Q000494:pharmacology; D002460:Cell Line; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D002999:Clone Cells / Q000187:drug effects / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D034281:Dynamins; D020558:GTP Phosphohydrolases / Q000494:pharmacology; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D011974:Receptors, LH / Q000819:agonists / Q000235:genetics* / Q000378:metabolism; D012694:Serine / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection; D014475:Uncoupling Agents / Q000494:pharmacology
D019393:Arrestin; D006063:Chorionic Gonadotropin; D011974:Receptors, LH; D014475:Uncoupling Agents; D012694:Serine; D000242:Cyclic AMP; D020558:GTP Phosphohydrolases; D034281:Dynamins; D000409:Alanine