Chemoattractant Receptors Activate Distinct Pathways for Chemotaxis and Secretion

Abstract

Human leukocyte chemoattractant receptors activate chemotactic and cytotoxic pathways to varying degrees and also activate different G-proteins depending on the receptor and the cell-type. To determine the relationship between G-protein usage and the biological and biochemical responses activated, receptors for the chemoattractants formyl peptides (FR), platelet-activating factor (PAFR), and leukotriene B 4 (BLTR) were transfected into RBL-2H3 cells. Pertussis toxin (Ptx) served as a G␣ i inhibitor. These receptors were chosen to represent the spectrum of G i usage as Ptx had differential effects on their ability to induce calcium mobilization, phosphoinositide hydrolysis, and exocytosis with complete inhibition of all responses by FR, intermediate effects on BLTR, and little effect on PAFR. Ptx did not affect ligand-induced phosphorylation of PAFR and BLTR but inhibited phosphorylation of FR. In contrast, chemotaxis to formylmethionylleucylphenylalanine, leukotriene B 4 , and platelet-activating factor was completely blocked by Ptx. Wortmannin, a phosphotidylinositol 3-kinase inhibitor, also completely blocked ligand-induced chemotaxis by all receptors but did not affect calcium mobilization or phosphoinositide hydrolysis; however, it partially blocked the exocytosis response to formylmethionylleucylphenylalanine and the platelet-activating factor. Membrane ruffling and pseudopod extension via the BLTR was also completely inhibited by both Ptx and wortmannin. These data suggest that of the chemoattractant receptors studied, G-protein usage varies with FR being totally dependent on G i , whereas BLTR and PAFR utilize both G i and a Ptxinsensitive G-protein. Both Ptx-sensitive and -insensitive G-protein usage can mediate the activation of phospholipase C, mobilization of intracellular calcium, and exocytosis by chemoattractant receptors. Chemotaxis, however, had an absolute requirement for a G i -mediated pathway.Migration of leukocytes to sites of inflammation is mediated via the activation of G-protein-coupled chemoattractant receptors (1, 2). Chemoattractants at low concentrations elicit shape change, pseudopod extension, and chemotaxis, and at higher doses; many of them also trigger degranulation and generation of superoxide anions (1, 3). Pathways leading to these activities have been shown to have different dose requirements, kinetics and regulation (4, 5), but the role of G-protein usage remains unknown.Formylpeptides (fMLP), 1 platelet-activating factor (PAF), and leukotriene B 4 (LTB 4 ) are potent chemoattractants for neutrophils and to varying degrees also activate exocytosis and generation of superoxide anions (3,4,6). These activities are mediated through G-protein-coupled receptors (FR, PAFR, and BLTR) (2, 7). G-protein usage of chemoattractant receptors was known to be different depending on cell types (1, 8 -10). Previous studies in RBL cells indicated that FR activated G i , whereas PAFR utilized both G i and a Ptx-insensitive G-protein to activate phosphoinositide hydrolysis, calcium mobilizatio...

Defective Macrophage Migration in Gαi2- but Not Gαi3-Deficient Mice

“…Within tissues, chemoattractants also direct the encounter between immune cells, thereby playing a regulatory role in immune homeostasis. Most chemoattractants, including chemokines, complement C5a, fMLP, leukotriene B 4 , and others (5)(6)(7)(8), signal through G protein-coupled receptors (GPCRs) that mediate their functions mainly via the heterotrimeric G i -proteins (9). Ligand binding to this class of GPCRs triggers Ga i subunits to exchange GTP for GDP, resulting in the dissociation of the Ga i subunit from Gbg heterodimers.…”

Platelet-activating Factor-induced Chemokine Gene Expression Requires NF-κB Activation and Ca2+/Calcineurin Signaling Pathways

“…PAF activates cell surface G protein-coupled receptors (GPCRs) to induce divergent biological functions (3)(4)(5)(6). PAF is a potent leukocyte chemoattractant (7), and it also induces degranulation (8,9), leukotriene C 4 generation (10,11), and chemokine gene expression in a wide variety of cells (12)(13)(14)(15)(16)(17). Although PAF-induced chemokine production appears to depend on the activation of transcription factor NF-B, a crucial transcription factor regulating the expression of many proinflammatory cytokines and immunoregulatory molecules (12,16,18,19), the early receptor-mediated signaling pathway that initiates this response has not been determined.…”

Distinct Roles of Receptor Phosphorylation, G Protein Usage, and Mitogen-activated Protein Kinase Activation on Platelet Activating Factor-induced Leukotriene C4 Generation and Chemokine Production

“…PAF activates cell surface G protein-coupled receptors (GPCRs) to induce divergent biological functions (3). PAF is a potent leukocyte chemoattractant (4) that also induces degranulation (5,6), leukotriene C 4 (LTC 4 ) generation (7,8), and chemokine gene expression in a wide variety of cells (9 -13). Although much has been learned regarding the signaling pathways involved in PAF-induced chemotaxis and degranulation (4,6,14), very little information is available on the mechanism by which PAF stimulates LTC 4 generation and chemokine production.…”

“…Unlike the C3a receptor, which couples to G i , the PAF receptor interacts with the G i and G q family of G proteins to induce distinct biological responses (4,23). In the present study, we sought to determine the roles of receptor phosphorylation, G protein usage, and ERK phosphorylation on PAF-induced LTC 4 generation and CCL2 production.…”