Chen Zhangguo, Dupré Denis J, Le Gouill Christian, Rola-Pleszczynski Marek, Stanková Jana
Immunology Division, Department of Pediatrics, Université de Sherbrooke, 3001 N 12th Avenue, Sherbrooke, Québec J1H 5N4, Canada.
J Biol Chem. 2002 Mar 1;277(9):7356-62. doi: 10.1074/jbc.M110058200. Epub 2001 Nov 29.
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.
与大多数G蛋白偶联受体一样，血小板活化因子受体（PAFR）反复受到激动剂刺激会导致其脱敏、隔离和内化。在本报告中，我们表明激动剂诱导的PAFR内化不依赖于G蛋白激活，而是依赖于抑制蛋白，并且涉及抑制蛋白与PAFR C末端有限区域的相互作用。在共转染的COS-7细胞中，抑制蛋白-2和抑制蛋白-3都可以与PAFR进行共免疫沉淀，并且PAFR的激动剂刺激会诱导抑制蛋白-2和抑制蛋白-3的转位。此外，抑制蛋白-2与PAFR的共表达增强了受体内化，而激动剂诱导的PAFR内化受到抑制蛋白-2的显性负突变体的抑制。编码受体C末端片段的小基因的共表达消除了PAF诱导的抑制蛋白转位并抑制了PAFR内化。使用C末端缺失突变体，我们确定抑制蛋白-2与受体的结合依赖于苏氨酸305和缬氨酸330之间的区域，因为抑制蛋白-2可以与突变体PAFRstop330进行免疫沉淀，但不能与PAFRstop305进行免疫沉淀。一致地，stop330可以介导激动剂诱导的抑制蛋白-2转位，而stop305则不能。另外两个C末端区域稍长的缺失突变体PAFRstop311和PAFRstop317也未能诱导抑制蛋白-2转位。最后，PAFR突变体Y293A在第七跨膜结构域的假定内化基序DPXXY中含有单个取代（我们已证明其能够内化但不能与G蛋白偶联），可以有效地诱导抑制蛋白转位。综上所述，我们的结果表明配体诱导的PAFR内化依赖于抑制蛋白，PAFR可以与抑制蛋白-2和-3都结合，并且它们的转位涉及与PAFR C末端318 - 330位残基区域的相互作用，但不依赖于G蛋白激活。
