Extracellular signal-regulated kinases (ERKs) play important physiological roles in proliferation, differentiation and gene expression. ERK5 is twice the size of ERK1/2, the amino-terminal half contains the kinase domain that shares the homology with ERK1/2 and TEY activation motif, whereas the carboxy-terminal half is unique. In this study, we examined the cross-talk mechanism between G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases, focusing on ERK1/2 and 5. The pretreatment of rat pheochromocytoma cells (PC12) with pertussis toxin (PTX) specifically enhanced epidermal growth factor (EGF)-induced ERK5 phosphorylation. In addition, lysophosphatidic acid (LPA) attenuated the EGF-induced ERK5 phosphorylation in LPA(1) receptor- and G(i/o)-dependent manners. On the other hand, LPA alone activated ERK1/2 via Gbetagamma subunits and Ras and potentiated EGF-induced ERK1/2 phosphorylation at late time points. These results suggest G(i/o) negatively regulates ERK5, while it positively regulates ERK1/2. LPA did not affect cAMP levels after EGF treatment, and the reagents promoting cAMP production such as forskolin and cholera toxin also attenuated the EGF-induced ERK5 phosphorylation, indicating that the inhibitory effect of LPA on ERK5 inhibition via G(i/o) is not due to inhibition of adenylyl cyclase by Galpha(i/o). However, the inhibitory effect of LPA on ERK5 was abolished in PC12 cells stably overexpressing C-terminus of GPCR kinase2 (GRK2), and overexpression of Gbeta(1) and gamma(2) subunits also suppressed ERK5 phosphorylation by EGF. In response to LPA, Gbetagamma subunits interacted with EGF receptor in a time-dependent manner. These results strongly suggest that LPA negatively regulates the EGF-induced ERK5 phosphorylation through Gbetagamma subunits.