Ahamed Jasimuddin, Ali Hydar
Department of Pathology, University of Pennsylvania School of Dental Medicine, Philadelphia, Pennsylvania 19104, USA.
J Biol Chem. 2002 Jun 21;277(25):22685-91. doi: 10.1074/jbc.M110210200. Epub 2002 Apr 4.
Platelet activating factor (PAF) interacts with cell surface G protein-coupled receptors on leukocytes to induce degranulation, leukotriene C(4) (LTC(4)) generation, and chemokine CCL2 production. Using a basophilic leukemia RBL-2H3 cell line expressing wild-type PAF receptor (PAFR) and a phosphorylation-deficient mutant (mPAFR), we have previously demonstrated that receptor phosphorylation mediates desensitization of PAF-induced degranulation. Here, we sought to determine the role of receptor phosphorylation on PAF-induced LTC(4) generation and CCL2 production. We found that PAF caused a significantly enhanced LTC(4) generation in cells expressing mPAFR when compared with PAFR cells. In contrast, PAF-induced CCL2 production was greatly reduced in mPAFR cells. Pertussis toxin and U0126, which inhibit G(i) and p44/42 mitogen-activated protein kinase (ERK) activation, respectively, caused very little inhibition of PAF-induced CCL2 production (approximately 20% inhibition). In contrast, these inhibitors almost completely blocked both PAF-induced ERK phosphorylation and LTC(4) generation in PAFR cells. However, in mPAFR cells pertussis toxin only partially inhibited PAF-induced ERK phosphorylation. A Ca(2+)/calmodulin inhibitor had no effect on PAF-induced ERK phosphorylation in PAFR cells but completely blocked the response in mPAFR cells. These data demonstrate that receptor phosphorylation, which serves to desensitize PAF-induced LTC(4) generation, is required for chemokine CCL2 production. They also indicate a previously unrecognized selectivity in G protein usage and ERK activation for PAF-induced responses. Whereas PAF-induced CCL2 production is, in large part, mediated independently of G(i) activation or ERK phosphorylation, LTC(4) generation requires ERK phosphorylation, which is mediated by different G proteins depending on the phosphorylation status of the receptor.
血小板活化因子（PAF）与白细胞表面的G蛋白偶联受体相互作用，以诱导脱颗粒、白三烯C4（LTC4）生成和趋化因子CCL2产生。利用表达野生型PAF受体（PAFR）和磷酸化缺陷型突变体（mPAFR）的嗜碱性白血病RBL - 2H3细胞系，我们先前已证明受体磷酸化介导PAF诱导的脱颗粒脱敏。在此，我们试图确定受体磷酸化对PAF诱导的LTC4生成和CCL2产生的作用。我们发现，与PAFR细胞相比，PAF在表达mPAFR的细胞中引起LTC4生成显著增强。相反，PAF诱导的CCL2产生在mPAFR细胞中大大减少。百日咳毒素和U0126分别抑制G(i)和p44/42丝裂原活化蛋白激酶（ERK）激活，对PAF诱导的CCL2产生几乎没有抑制作用（约20%抑制）。相反，这些抑制剂几乎完全阻断了PAF诱导的PAFR细胞中ERK磷酸化和LTC4生成。然而，在mPAFR细胞中，百日咳毒素仅部分抑制PAF诱导的ERK磷酸化。一种Ca(2+)/钙调蛋白抑制剂对PAFR细胞中PAF诱导的ERK磷酸化没有影响，但完全阻断了mPAFR细胞中的反应。这些数据表明，受体磷酸化虽然使PAF诱导的LTC4生成脱敏，但却是趋化因子CCL2产生所必需的。它们还表明在PAF诱导的反应中，G蛋白使用和ERK激活存在先前未被认识到的选择性。虽然PAF诱导的CCL2产生在很大程度上独立于G(i)激活或ERK磷酸化介导，但LTC4生成需要ERK磷酸化，这取决于受体的磷酸化状态由不同的G蛋白介导。
