Vines Charlotte M, Xue Mei, Maestas Diane C, Cimino Daniel F, Prossnitz Eric R
Department of Cell Biology and Physiology, University of New Mexico Health Science Center, Albuquerque, NM 87131, USA.
J Immunol. 2002 Dec 15;169(12):6760-6. doi: 10.4049/jimmunol.169.12.6760.
One of the major functions of the N-formyl peptide receptor (FPR) is to mediate leukocyte degranulation. Phosphorylation of the C-terminal domain of the FPR is required for receptor internalization and desensitization. Although arrestins mediate phosphorylation-dependent desensitization, internalization, and initiation of novel signaling cascades for a number of G protein-coupled receptors, their roles in FPR regulation and signaling remain unclear. CXCR1-mediated degranulation of RBL-2H3 cells is promoted by arrestin binding. To determine whether receptor phosphorylation or arrestin binding is required to promote FPR-mediated degranulation, we used RBL-2H3 cells stably transfected with either the wild-type FPR or a mutant form, DeltaST, which is incapable of undergoing ligand-stimulated phosphorylation. We observed that stimulation of wild-type FPR resulted in very low levels of degranulation compared with that mediated by cross-linking of the Fc(epsilon)RI receptor. Stimulation of the DeltaST mutant, however, resulted in levels of degranulation comparable to those of the Fc(epsilon)RI receptor, demonstrating that neither receptor phosphorylation nor arrestin binding was necessary to initiate FPR-mediated degranulation. Degranulation initiated by the DeltaST mutant was proportional to the level of active cell surface receptor, suggesting that either receptor internalization or desensitization may be responsible for terminating degranulation of the wild-type FPR. To distinguish between these possibilities, we used a partially phosphorylation-deficient mutant of the FPR that can undergo internalization, but not desensitization. Degranulation by this mutant FPR was indistinguishable from that of the DeltaST mutant, indicating that FPR phosphorylation or binding of arrestin but not internalization terminates the degranulation response.
N-甲酰肽受体（FPR）的主要功能之一是介导白细胞脱颗粒。FPR C末端结构域的磷酸化是受体内化和脱敏所必需的。尽管抑制蛋白介导许多G蛋白偶联受体的磷酸化依赖性脱敏、内化及新信号级联反应的起始，但它们在FPR调节和信号传导中的作用仍不清楚。抑制蛋白结合可促进CXCR1介导的RBL-2H3细胞脱颗粒。为了确定促进FPR介导的脱颗粒是否需要受体磷酸化或抑制蛋白结合，我们使用稳定转染野生型FPR或突变形式DeltaST的RBL-2H3细胞，DeltaST不能进行配体刺激的磷酸化。我们观察到，与Fc(ε)RI受体交联介导的脱颗粒相比，野生型FPR的刺激导致脱颗粒水平非常低。然而，DeltaST突变体的刺激导致脱颗粒水平与Fc(ε)RI受体相当，表明受体磷酸化和抑制蛋白结合都不是启动FPR介导的脱颗粒所必需的。DeltaST突变体引发的脱颗粒与活性细胞表面受体水平成正比，这表明受体内化或脱敏可能负责终止野生型FPR的脱颗粒。为了区分这些可能性，我们使用了一种FPR的部分磷酸化缺陷突变体，它可以进行内化，但不能脱敏。这种突变型FPR的脱颗粒与DeltaST突变体的脱颗粒没有区别，表明FPR磷酸化或抑制蛋白结合而非内化终止了脱颗粒反应。
