S by activating subsets of G proteins. COS-7 cells have already been extensively used to characterize EGFR transactivation [15]. To examine which EP receptors could activate EGFR and whether metalloproteinase activity was expected, we expressed each of the 4 EP receptors in COS-7 cells, treated the cells with PGE2, and then measured TBK1 MedChemExpress phosphorylation of Akt at Ser473 in the presence of either an EGFR inhibitor (AG1478) or maybe a broad spectrum metalloproteinase inhibitor (PLK1 Synonyms GM6001, Ilomistat). We located that Akt was not phosphorylated in COS-7 cells transfected with all the empty vector (Fig 2A). Nor was it phosphorylated in cells expressing EP1. Nonetheless, Akt was phosphorylated in cells expressing EP2, EP3, or EP4 (Fig. 2A). Additionally, the inhibitors had distinct effects on this phosphorylation. In cells expressing EP2, Akt phosphorylation was fully inhibited by each AG1478 and GM6001, indicating that activation of Akt by means of EP2 expected each EGFR and metalloproteinase activity, respectively. This indicated that EP2 transactivated EGFR by means of the well-defined pathway involving activation of a metalloproteinase and subsequent release from the development issue ligands that bind EGFR. EP3 also triggered Akt phosphorylation, but this was only partially inhibited by either AG1478 or GM6001, indicating that EP3 caused Akt phosphorylation by metalloproteinase and EGFR-dependent and -independent mechanisms. Finally, Akt was phosphorylated in cells expressing EP4, but this was not inhibited by either AG1478 or GM6001. We also examined phosphorylation of Akt at Thr308 and identified related outcomes (not shown). Moreover, we measured ERK1/2 phosphorylation and discovered that PGE2 triggered ERK1/2 phosphorylation that was not drastically affected by either AG1478 or GM6001, indicating that ERK1/2 activation predominantly occurs directly by means of the EP receptors in lieu of by way of EGFR. We conclude that EP2 and EP3 can activate Akt via a metalloproteinase and EGFR. Some EP receptors couple to Gi subunits, that are sensitive to pertussis toxin. To test the value of Gi subunits, we treated HEK293 cells with pertussis toxin and after that examined PGE2-induced ERK1/2 and Akt activation. HEK293 cells express mRNA for all 4 EP receptors (information not shown). We discovered that pertussis toxin completely inhibited PGE2-induced Akt phosphorylation (Fig. 2C), indicating that in HEK293 cells, Gi subunits are essential. The robust, EGFR-independent activation of Akt in cells expressing EP4 was not surprising simply because G protein-coupled receptors are known to activate phosphatidylinositol 3-kinases, and consequently Akt, by mechanisms that don’t involve transactivation of EGFR [19]. However,NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptCell Signal. Author manuscript; out there in PMC 2009 May 13.Al-Salihi et al.Pagewe regarded the possibility that EP4 could possibly have transactivated EGFR, but that this was masked by EGFR-independent Akt phosphorylation. To additional directly assess EGFR activation, we co-expressed EGFR and also the EP receptors in COS-7 cells and after that assayed the status of EGFR using a phosphorylation-specific antibody. Constant together with the final results in Fig. 2A, we discovered that PGE2 did not cause EGFR phosphorylation in cells expressing EP1, but did result in EGFR phosphorylation in cells expressing EP2 or EP3 (Fig. 2D). Surprisingly, EGFR was also phosphorylated in cells expressing EP4 (Fig. 2D). Using scanning densitometry to quantify the Western blots, we found statis.