Activation of the mitogen-activated protein kinases Erk1/2 by erythropoietin receptor via a Gi protein βγ-subunit-initiated pathway

We have recently shown that a heterotrimeric G(i) protein is coupled to the erythropoietin (Epo) receptor. The G(i) protein constitutively associates in its heterotrimeric form with the intracellular domain of Epo receptor (EpoR). After Epo stimulation G(i) is released from the receptor and activated. In the present study we have investigated the functional role of the heterotrimeric G(i) protein bound to EpoR. In Chinese hamster ovary cells expressing EpoR, the G(i) inhibitor pertussis toxin blocked mitogen-activated protein kinase (MAPK) Erk1/2 activation induced by Epo. Epo-dependent MAPK activation was also sensitive to the Gbetagamma competitive inhibitor betaARK1-ct (C-terminal fragment of the beta-adrenergic receptor kinase), to the Ras dominant negative mutant RasN17, and to the phosphoinositide 3-kinase (PI3K) inhibitor LY 294002. A region of 7 amino acids (469-475) in the C-terminal end of EpoR was shown to be required for G(i) binding to EpoR in vivo. Deletion of this region in EpoR abolished both MAPK and PI3K activation in response to Epo. We conclude that in Chinese hamster ovary cells, Epo activates MAPK via a novel pathway dependant on G(i) association to EpoR, Gbetagamma subunit, Ras, and PI3K. The tyrosine kinase Jak2 also contributes to this new pathway, more likely downstream of betagamma and upstream of Ras and PI3K. This pathway is similar to the best characterized pathway used by seven transmembrane receptors coupled to G(i) to activate MAPK and may cooperate with other described Epo-dependent MAPK activation pathways in hematopoietic cells.