S by activating subsets of G proteins. COS-7 cells have been extensively made use of to characterize EGFR transactivation [15]. To examine which EP receptors could activate EGFR and regardless of whether metalloproteinase Integrin Associated Protein/CD47 Proteins Biological Activity activity was necessary, we expressed every with the 4 EP receptors in COS-7 cells, treated the cells with PGE2, and then measured phosphorylation of Akt at Ser473 within the presence of either an EGFR inhibitor (AG1478) or even a broad spectrum metalloproteinase inhibitor (GM6001, Ilomistat). We identified that Akt was not phosphorylated in COS-7 cells transfected together with the empty vector (Fig 2A). Nor was it phosphorylated in cells expressing EP1. On the other hand, Akt was phosphorylated in cells expressing EP2, EP3, or EP4 (Fig. 2A). Furthermore, the inhibitors had diverse effects on this phosphorylation. In cells expressing EP2, Akt phosphorylation was absolutely inhibited by both AG1478 and GM6001, indicating that activation of Akt by means of EP2 essential both EGFR and metalloproteinase activity, respectively. This indicated that EP2 transactivated EGFR through the well-defined pathway involving activation of a metalloproteinase and subsequent release from the growth factor ligands that bind EGFR. EP3 also caused Akt phosphorylation, but this was only partially inhibited by either AG1478 or GM6001, indicating that EP3 triggered Akt phosphorylation by metalloproteinase and EGFR-dependent and -independent mechanisms. Ultimately, 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 located equivalent benefits (not shown). Also, we measured ERK1/2 phosphorylation and found that PGE2 caused ERK1/2 phosphorylation that was not significantly affected by either AG1478 or GM6001, indicating that ERK1/2 activation predominantly happens straight by means of the EP receptors as opposed to through EGFR. We conclude that EP2 and EP3 can activate Akt through a metalloproteinase and EGFR. Some EP receptors couple to Gi subunits, which are sensitive to pertussis toxin. To test the importance of Gi subunits, we treated HEK293 cells with pertussis toxin and then examined PGE2-induced ERK1/2 and Akt activation. HEK293 cells express mRNA for all four EP receptors (information not shown). We found that pertussis toxin totally inhibited PGE2-induced Akt phosphorylation (Fig. 2C), indicating that in HEK293 cells, Gi subunits are important. The robust, EGFR-independent activation of Akt in cells expressing EP4 was not surprising mainly because G protein-coupled receptors are known to activate phosphatidylinositol 3-kinases, and consequently Akt, by mechanisms that never involve transactivation of EGFR [19]. On the other hand,NIH-PA Author IDO Proteins Accession Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptCell Signal. Author manuscript; accessible in PMC 2009 May perhaps 13.Al-Salihi et al.Pagewe regarded as the possibility that EP4 could possibly have transactivated EGFR, but that this was masked by EGFR-independent Akt phosphorylation. To more straight assess EGFR activation, we co-expressed EGFR and the EP receptors in COS-7 cells and then assayed the status of EGFR using a phosphorylation-specific antibody. Consistent using the benefits in Fig. 2A, we discovered that PGE2 didn’t result in 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). Working with scanning densitometry to quantify the Western blots, we found statis.