Department of Biology, Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, Durham, NC 27707, USA.
J Immunol. 2012 Sep 15;189(6):2824-32. doi: 10.4049/jimmunol.1201114. Epub 2012 Aug 6.
The chemokine receptors, CXCR1 and CXCR2, couple to Gαi to induce leukocyte recruitment and activation at sites of inflammation. Upon activation by CXCL8, these receptors become phosphorylated, desensitized, and internalized. In this study, we investigated the role of different G protein-coupled receptor kinases (GRKs) in CXCR1- and CXCR2-mediated cellular functions. To that end, short hairpin RNA was used to inhibit GRK2, 3, 5, and 6 in RBL-2H3 cells stably expressing CXCR1 or CXCR2, and CXCL8-mediated receptor activation and regulation were assessed. Inhibition of GRK2 and GRK6 increased CXCR1 and CXCR2 resistance to phosphorylation, desensitization, and internalization, respectively, and enhanced CXCL8-induced phosphoinositide hydrolysis and exocytosis in vitro. GRK2 depletion diminished CXCR1-induced ERK1/2 phosphorylation but had no effect on CXCR2-induced ERK1/2 phosphorylation. GRK6 depletion had no significant effect on CXCR1 function. However, peritoneal neutrophils from mice deficient in GRK6 (GRK6(-/-)) displayed an increase in CXCR2-mediated G protein activation but in vitro exhibited a decrease in chemotaxis, receptor desensitization, and internalization relative to wild-type (GRK6(+/+)) cells. In contrast, neutrophil recruitment in vivo in GRK6(-/-) mice was increased in response to delivery of CXCL1 through the air pouch model. In a wound-closure assay, GRK6(-/-) mice showed enhanced myeloperoxidase activity, suggesting enhanced neutrophil recruitment, and faster wound closure compared with GRK6(+/+) animals. Taken together, the results indicate that CXCR1 and CXCR2 couple to distinct GRK isoforms to mediate and regulate inflammatory responses. CXCR1 predominantly couples to GRK2, whereas CXCR2 interacts with GRK6 to negatively regulate receptor sensitization and trafficking, thus affecting cell signaling and angiogenesis.
趋化因子受体 CXCR1 和 CXCR2 通过与 Gαi 偶联在炎症部位诱导白细胞募集和激活。在被 CXCL8 激活后，这些受体被磷酸化、脱敏和内化。在这项研究中，我们研究了不同的 G 蛋白偶联受体激酶（GRK）在 CXCR1 和 CXCR2 介导的细胞功能中的作用。为此，使用短发夹 RNA 抑制 RBL-2H3 细胞中稳定表达的 CXCR1 或 CXCR2 中的 GRK2、3、5 和 6，评估 CXCL8 介导的受体激活和调节。GRK2 和 GRK6 的抑制分别增加了 CXCR1 和 CXCR2 对磷酸化、脱敏和内化的抗性，并增强了体外的 CXCL8 诱导的磷酸肌醇水解和胞吐作用。GRK2 耗竭减少了 CXCR1 诱导的 ERK1/2 磷酸化，但对 CXCR2 诱导的 ERK1/2 磷酸化没有影响。GRK6 耗竭对 CXCR1 功能没有显著影响。然而，GRK6(-/-) 小鼠的腹腔中性粒细胞显示出 CXCR2 介导的 G 蛋白激活增加，但体外趋化性、受体脱敏和内化减少与野生型（GRK6(+/+)）细胞相比。相比之下，在通过气囊中模型递送 CXCL1 后，GRK6(-/-) 小鼠体内的中性粒细胞募集增加。在伤口闭合测定中，GRK6(-/-) 小鼠显示出增强的髓过氧化物酶活性，表明中性粒细胞募集增强，与 GRK6(+/+) 动物相比，伤口闭合更快。总之，结果表明 CXCR1 和 CXCR2 通过与不同的 GRK 同工型偶联来介导和调节炎症反应。CXCR1 主要与 GRK2 偶联，而 CXCR2 与 GRK6 相互作用以负调节受体敏化和运输，从而影响细胞信号转导和血管生成。
