Role of guanine nucleotide exchange factor P-Rex-2b in sphingosine 1-phosphate-induced Rac1 activation and cell migration in endothelial cells

Endothelial cell (EC) migration has an important role in angiogenesis. Sphingosine-1 phosphate (S1P) stimulates EC migration via activation of Gi proteins. In this study, we characterized a mouse guanine nucleotide exchange factor (GEF) P-Rex2b for its regulation by Gβγ and PI3K and its role in S1P-induced Rac1 activation and cell migration in ECs. We found that co-expression of Gβγ or an active form of PI3K (PI3KAC) with P-Rex2b increased the SRE.Luciferase (SRE.L) reporter gene activity that can be stimulated by the Rho family of small GTPases including Rac1. Co-expression with P-Rex2b of Gβγ and PI3KAC or wild type PI3Kγ that can be activated by Gβγ led to further increases in the reporter gene activity. Together with the finding that co-expression of Gβγ and/or PI3KAC increased the levels of active Rac1, we conclude that P-Rex2b is a Rac GEF that can be regulated by Gβγ and PI3K. Additionally, we demonstrated that Gβγ interacted with P-Rex2b, probably through P-Rex2b sequences at the PH domain and that the DEP and PDZ domains of P-Rex2b exerted an inhibitory effect on P-Rex2b's activity because their deletion increased the SER.L reporter gene activity. Furthermore, we found that P-Rex2b is involved in S1P-induced Rac1 activation and cell migration in ECs because siRNA-mediated suppression of P-Rex2b expression in ECs-diminished Rac1 activation and cell migration in response to S1P. Therefore, P-Rex2b is a physiologically significant Rac1 GEF that has an important role in the regulation of EC migration.