Heterotrimeric G proteins physically associated with the lipopolysaccharide receptor CD14 modulate both in vivo and in vitro responses to lipopolysaccharide.

Abstract

Septic shock induced by lipopolysaccharide (LPS) triggering of cytokine production from monocytes/macrophages is a major cause of morbidity and mortality. The major monocyte/macrophage LPS receptor is the glycosylphosphatidylinositol (GPI)-anchored glycoprotein CD14. Here we demonstrate that CD14 coimmunoprecipitates with G i /G o heterotrimeric G proteins. Furthermore, we demonstrate that heterotrimeric G proteins specifically regulate CD14-mediated, LPS-induced mitogen-activated protein kinase (MAPK) activation and cytokine production in normal human monocytes and cultured cells. We report here that a G protein binding peptide protects rats from LPS-induced mortality, suggesting a functional linkage between a GPIanchored receptor and the intracellular signaling molecules with which it is physically associated. ( J. Clin. Invest. 1998 .

Mastoparan, a G Protein Agonist Peptide, Differentially Modulates TLR4- and TLR2-Mediated Signaling in Human Endothelial Cells and Murine Macrophages

“…The findings presented herein confirm the findings of Solomon et al (33) and extend them in several significant ways. First, given that CD14 and G proteins were found to coimmunoprecipitate (33), one might predict that cells that lack mCD14 would not be sensitive to pharmacologic disruption of G protein signaling. However, our findings that mastoparan inhibited most facets of LPS signaling in CD14-deficient HMEC suggest that the G proteins are not physically associated with CD14.…”

“…Fig. 3, C and D, showed the inhibitory effect of mastoparan on TLR4-mediated p38 phosphorylation and kinase activity in HMEC, consistent with previous reports in human monocytes (33). To confirm and extend these findings in murine macrophages, the RAW 264.7 cell line was also assessed for its ability to respond to LPS in the absence or presence of mastoparan by measuring IRAK-1-associated kinase activity.…”

“…2B) was dose-dependent. As a positive control, we also confirmed in the murine macrophage cell line, RAW 264.7, the previously reported inhibitory effect of mastoparan on LPS-induced cytokine release by human monocytes/PBMC (33). Fig.…”

“…For example, in studies reported by Zhang and Morrison (39,40), PT pretreatment of peritoneal exudate macrophages resulted in inhibition of LPS-induced NO release, while TNF-␣ secretion was augmented, an observation that we have confirmed independently (A. Lentschat, data not shown). More recently, Solomon et al (33) failed to observe PT-induced inhibition of LPS-induced signaling in human monocytes, while mastoparan pretreatment uniformly inhibited LPS-induced responses and afforded significant protection in vivo in a model of LPS-induced shock in lead acetatesensitized rats. Ferlito et al (62) used Chinese hamster ovary cells stably transfected with CD14 and found that PT blocked LPSinduced p38 phosphorylation, but not I B␣ degradation, while mastoparan synergized with LPS for p38 phosphorylation.…”

“…Ligand binding of GPCRs activates the MAPKs, JNK and p38 (28 -31). Coimmunoprecipitation experiments have revealed that src kinases and G i /G 0 heterotrimeric G proteins also associate with CD14 in human monocyte lysates (32,33). Lastly, some studies have found that treatment of cells with Bordetella pertussis toxin (PT), a G protein inhibitor (34), or mastoparan, a peptide derived from wasp venom that activates G proteins (35,36), modulate LPS-induced B cell mitogenesis, and macrophage cytokine and NO production, and p38 phosphorylation (33,(37)(38)(39)(40), although in some studies, PT exerted either no effect or even enhanced LPSmediated responses (e.g., Refs.…”