Role of cytoplasmic tail phosphorylation sites of platelet-activating factor receptor in agonist-induced desensitization.
The platelet-activating factor (PAF) receptor couples with multiple signaling pathways such as activation of phospholipase C, phospholipase A2, and mitogen-activated protein kinase and the inhibition of adenylate cyclase. The PAF-induced signals are attenuated by repetitive or long standing applications of the agonist (homologous desensitization). To investigate mechanisms underlying the agonist-induced desensitization, we constructed mutant forms of the cloned guinea pig PAF receptor and stably expressed them in Chinese hamster ovary cells. The cells expressing the wild type receptor transiently activated phospholipase C in response to PAF. Intracellular inositol 1,4,5-trisphosphate level and intracellular Ca2+ concentration reached the maximal levels within 20 s and returned to the basal levels in several minutes, even in the continuous presence of the ligand. In contrast, a truncated PAF receptor lacking the carboxyl-terminal cytoplasmic tail induced sustained elevations of inositol 1,4,5-trisphosphate and intracellular Ca2+ concentrations. Similar findings were noted in another mutant, in which the Ser/Thr residues in the carboxyl-terminal tail were substituted with Ala. Both mutant PAF receptors more potently activated the other signals (mitogen-activated protein kinase kinase, arachidonate release, and inhibition of adenylate cyclase) than did the wild type receptor. Thus, while the carboxyl-terminal cytoplasmic tail of the PAF receptor is not required for the forward activation of multiple signals, it does have a critical role for signal attenuation induced by the agonist through phosphate accepters. We also noted that the synthetic peptide of the PAF receptor carboxyl-terminal tail was strongly phosphorylated by the recombinant beta-adrenergic receptor kinase 1, suggesting that it or its relatives might be involved in PAF receptor phosphorylation and homologous desensitization.
Takano
T
T
Department of Biochemistry, Faculty of Medicine, University of Tokyo, Japan.
Honda
Z
Z
Sakanaka
C
C
Izumi
T
T
Kameyama
K
K
Haga
K
K
Haga
T
T
Kurokawa
K
K
Shimizu
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D006168:Guinea Pigs; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D010766:Phosphorylation; D010972:Platelet Activating Factor / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D015398:Signal Transduction; D013917:Thrombin / Q000378:metabolism; D051552:beta-Adrenergic Receptor Kinases
D009838:Oligodeoxyribonucleotides; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C039351:platelet activating factor receptor; D015544:Inositol 1,4,5-Trisphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D013917:Thrombin; D002118:Calcium