Agonist-induced sequestration, recycling, and resensitization of platelet-activating factor receptor. Role of cytoplasmic tail phosphorylation in each process.
Agonist-induced sequestration, recycling, and resensitization of platelet-activating factor (PAF) receptor were characterized in transfected Chinese hamster ovary cells. Exposure of the cells to PAF led to rapid sequestration of the receptors into the intracellular compartment and desensitization of the response to PAF. The sequestration was inhibited by pretreatments that perturbed the clathrin-mediated pathway. Subsequent removal of PAF by washing with receptor antagonists led to rapid recycling of the sequestered receptors to the cell surface accompanied by resensitization to PAF. To evaluate the potential role of phosphorylation in the receptor cytoplasmic tail during these processes, mutant receptors in which the tails were truncated or substituted, so as to lack serine/threonine residues, were created. PAF phosphorylated the wild-type receptor rapidly and strongly, but the mutants did not. The maximal extent of sequestration of each mutant was lower than that of the wild-type, and one of the substituted mutants showed no sequestration. Furthermore, the sequestration-defective mutant showed evidence of desensitization after agonist stimulation but not resensitization after agonist removal. Thus, agonist-induced phosphorylation of the cytoplasmic tail facilitates but is not essential for receptor sequestration, and sequestration/recycling appears important in receptor resensitization.
Ishii
I
I
Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo, Tokyo 113, Japan.
Saito
E
E
Izumi
T
T
Ui
M
M
Shimizu
T
T
D000595:Amino Acid Sequence; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002966:Clathrin / Q000378:metabolism; D006224:Cricetinae; D005492:Folic Acid / Q000378:metabolism; D006168:Guinea Pigs; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D008958:Models, Molecular; D009842:Oligopeptides; D010455:Peptides; D010766:Phosphorylation; D010972:Platelet Activating Factor / Q000494:pharmacology*; D010980:Platelet Membrane Glycoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000502:physiology*; D014176:Protein Biosynthesis; D017433:Protein Structure, Secondary; D011869:Radioligand Assay; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D014162:Transfection
D002966:Clathrin; D009842:Oligopeptides; D010455:Peptides; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; C039351:platelet activating factor receptor; D016718:Arachidonic Acid; D015544:Inositol 1,4,5-Trisphosphate; D005492:Folic Acid; C085706:FLAG peptide