Activation of the Mitogen-activated Protein Kinases Erk1/2 by Erythropoietin Receptor via a Gi Protein βγ-Subunit-initiated Pathway

Abstract

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 G␤␥ competitive inhibitor ␤ARK1-ct (C-terminal fragment of the ␤-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, G␤␥ subunit, Ras, and PI3K. The tyrosine kinase Jak2 also contributes to this new pathway, more likely downstream of ␤␥ 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.

Cardioprotective Effects of Erythropoietin in the Reperfused Ischemic Heart

“…7). This is actually similar to previous findings in CHO cells where EPO activation of ERK was G i -dependent (27). EPO also induces phosphorylation of Akt by 1.88 Ϯ 0.42-fold increase over basal (n ϭ 4, p Ͻ 0.05) (Fig.…”

“…In recent studies performed to define EPO-mediated regulation of the ERK cascade, several different pathways have been identified that can ultimately lead to ERK activation including PI3K, which may use an atypical protein kinase C for EPOinduced ERK activation in CHO-K1 cells (27). All ERK pathways appear to be initiated through effectors binding to phosphorylated tyrosines located in the intracellular domain of the EPO-R (35).…”

Core erythropoietin receptor signals for late erythroblast development

“…56 In contrast, the presently observed strong activation of ERKs via EpoR-HM in primary erythroblasts suggests that PY sites may be nonessential for ERK activation. Candidate factors that might couple EpoR-HM (and perhaps the wt-EpoR) to ERKs are presently undefined, but G-proteins as well as PKCs stimulate MAPK modules 57,58 and represent 2 sets of potentially Epo-regulated candidate effectors. Based on the substantial in vivo activity exerted by EpoR-HM, such Epo-regulated pathways should be of interest to define.…”

Different Pathways for Activation of Extracellular Signal-Regulated Kinase through Thromboxane A2 Receptor Isoforms

“…33,34) The inovolvement of G i in phosphorylation of ERK1/2 was reported in the case of other GPCRs, such as dopamine D 2 receptor 35) and erythropoietin receptor. 36) In the case of TP, however, there are distinct reports about the involvement of G i in the phosphorylation of ERK1/2. Although it was shown that G i participated in TPmediated ERK1/2 phosphorylation in human bladder cancer cells, 37) Miggin and Kinsella 26) reported that G i did not participate in TP-mediated ERK1/2 phosphorylation in human uterine smooth muscle cells.…”