Activation of the mitogen-activated protein kinases Erk1/2 by erythropoietin receptor via a G_i 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 Gβγ competitive inhibitor βARK1-ct (C-terminal fragment of the β-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, Gβγ subunit, Ras, and PI3K. The tyrosine kinase Jak2 also contributes to this new pathway, more likely downstream of βγ 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.