Agonist-induced internalization of the platelet-activating factor receptor is dependent on arrestins but independent of G-protein activation. Role of the C terminus and the (D/N)PXXY motif.
As with most G-protein-coupled receptors, repeated agonist stimulation of the platelet-activating factor receptor (PAFR) results in its desensitization, sequestration, and internalization. In this report, we show that agonist-induced PAFR internalization is independent of G-protein activation but is dependent on arrestins and involves the interaction of arrestins with a limited region of the PAFR C terminus. In cotransfected COS-7 cells, both arrestin-2 and arrestin-3 could be coimmunoprecipitated with PAFR, and agonist stimulation of PAFR induced the translocation of both arrestin-2 and arrestin-3. Furthermore, coexpression of arrestin-2 with PAFR potentiated receptor internalization, whereas agonist-induced PAFR internalization was inhibited by a dominant negative mutant of arrestin-2. The coexpression of a minigene encoding the C-terminal segment of the receptor abolished PAF-induced arrestin translocation and inhibited PAFR internalization. Using C terminus deletion mutants, we determined that the association of arrestin-2 with the receptor was dependent on the region between threonine 305 and valine 330 because arrestin-2 could be immunoprecipitated with the mutant PAFRstop330 but not PAFRstop305. Consistently, stop330 could mediate agonist-induced arrestin-2 translocation, whereas stop305 could not. Two other deletion mutants with slightly longer regions of the C terminus, PAFRstop311 and PAFRstop317, also failed to induce arrestin-2 translocation. Finally, the PAFR mutant Y293A, containing a single substitution in the putative internalization motif DPXXY in the seventh transmembrane domain (which we had shown to be able to internalize but not to couple to G-proteins) could efficiently induce arrestin translocation. Taken together, our results indicate that ligand-induced PAFR internalization is dependent on arrestins, that PAFR can associate with both arrestin-2 and -3, and that their translocation involves interaction with the region of residues 318-330 in the PAFR C terminus but is independent of G-protein activation.
Chen
Zhangguo
Z
Immunology Division, Department of Pediatrics, Université de Sherbrooke, 3001 N 12th Avenue, Sherbrooke, Québec J1H 5N4, Canada.
Dupré
Denis J
DJ
Le Gouill
Christian
C
Rola-Pleszczynski
Marek
M
Stanková
Jana
J
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D019393:Arrestin / Q000737:chemistry* / Q000378:metabolism*; D019390:Arrestins / Q000378:metabolism; D001667:Binding, Competitive; D016466:CHO Cells; D019556:COS Cells; D002460:Cell Line; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D000939:Epitopes; D005434:Flow Cytometry; D019204:GTP-Binding Proteins / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009154:Mutation; D010750:Phosphoproteins / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000819:agonists* / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D013912:Threonine / Q000737:chemistry; D014162:Transfection; D014633:Valine / Q000737:chemistry
D019393:Arrestin; D019390:Arrestins; D000939:Epitopes; D008164:Luminescent Proteins; D010750:Phosphoproteins; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C082333:arrestin3; C039351:platelet activating factor receptor; D049452:Green Fluorescent Proteins; D013912:Threonine; D019204:GTP-Binding Proteins; D014633:Valine