Gs/cAMP signaling is important for acidosis/GPR4- induced HUVEC cell adhesion GPCRs are cell surface receptors that transduce signals mainly through G protein coupling [46,47,48], but G protein-indepen- dent pathways have also been reported [49]. Some GPCRs, particularly those with a long amino-terminal extracellular domain, can directly function as adhesion molecules to increase cell adhesion [50]. GPR4 does not have a long amino-terminal extracellular domain and thus is unlikely to serve as an adhesion molecule per se. Furthermore, we observed that the mRNA level of GPR4 was actually decreased by ,50% in HUVEC/Vector cells upon acidosis treatment for 5 h (Fig. S2). This may represent a regulatory mechanism to down-regulate GPR4 signaling after its activation by acidosis, and the results also indicate that GPR4 itself may not directly act as an adhesion molecule to increase HUVEC adhesiveness. To elucidate GPR4 downstream pathways and to further rule out the possibility that GPR4 mediates cell adhesion by functioning as an adhesion molecule, we generated a GPR4 mutant with the arginine 115 to alanine (R115A) mutation in the DRY motif, which is located at the second intracellular loop and important for G protein signaling [38,40]. The GPR4 R115A mutant was transduced into HUVEC cells (designated as Acidosis/GPR4 and Endothelial Cell Adhesion HUVEC/GPR4 R115A cells) and the expression of the GPR4 R115A mutant was at the comparable level as that of GPR4 in HUVEC/GPR4 cells (data not shown). To examine the G protein signaling of the GPR4 R115A mutant, acidosis/GPR4-induced cAMP production was measured. Acidic pH stimulated the production of cAMP by about 7-fold in HUVEC/GPR4 cells but not in the HUVEC/GPR4 R115A cells (Fig. 2A), suggesting that the R115A mutant is defective in G protein signaling. Upon the treatment of pH 6.4, HUVECs overexpressing the wild-type GPR4 showed a ,10-fold increase of adhesion with U937 cells. However, overexpression of the GPR4 R115A mutant did not augment HUVEC cell adhesion at acidic pH (Fig. 2B). These results indicate that G protein signaling is indispensable for acidosis/GPR4-mediated endothelial cell adhesion. Since GPR4 was co-expressed with a bicistronic GFP marker, we examined whether the adhesiveness of HUVEC cells is linearly correlated with the intensity of GFP signal, an indicator of GPR4 expression. Overall, the GFP signal was heterogeneous and relatively low in the transduced HUVECs (Fig. S3). Some HUVEC cells with strong GFP signals did not always showed strong adhesion with U937 cells and, on the other hand, some HUVEC cells with low to medium GFP signals exhibited high Figure 4. Epac is important for HUVEC adhesion induced by acidosis/GPR4. (A) HUVEC/GPR4 cells were pretreated with vehicle or H-89 (10 mM) for 1 h. Cells were then treated with indicated pH media containing vehicle or 10 mM H-89 for 5 to 15 h (overnight), and the cell adhesion assay was performed as described under ‘‘Materials and Methods’’. ns, not significant (P.0.05). (B) HUVEC/Vector or HUVEC/GPR4 cells were stably transduced with the myc-tagged Epac dominant-negative construct N-Epac or the control vector pQCXIP. Total proteins were extracted from HUVEC cells and subject to Western blot analysis for myc-N-Epac with anti-myc antibodies. GAPDH was used as a loading control. (C) HUVEC/Vector or HUVEC/GPR4 cells were stably transduced with the dominant-negative N-Epac or the control vector pQCXIP. Cells were treated with indicated pH media for 5 or 15 h and then the cell adhesion assay was performed. **, P,0.01; ***, P,0.001. (D) HUVEC/GPR4 cells were treated with indicated pH media containing vehicle or 100 mM 8-CPT-2Me-cAMP for 15 h, and then the cell adhesion assay was performed. All the above results are representative of two or more independent experiments. Error bars are the mean 6 SEM. doi:10.1371/journal.pone.0027586.g004 PLoS ONE | www.plosone.org 7 November 2011 | Volume 6 | Issue 11 | e27586