As discussed above, estrogen initiates multiple intracellular signaling cascades. Although classical estrogen receptors have been demonstrated to be capable of mediating many of these responses, the signaling capabilities of GPR30 in response to estrogen have just begun to be described. Estrogen-mediated GPR30-dependent activation of the MAP kinase Erk1/2 via EGFR transactivation was first described in 2000 (Filardo et al., 2000
). Subsequently, adenylyl cyclase activation by GPR30 has also been demonstrated (Filardo et al., 2002
; Thomas et al., 2005
). However, following these studies, Levin and colleagues questioned these original conclusions in a paper where they reported similar EGFR transactivation and Erk activation mediated by estrogen through ERα (Razandi et al., 2003
). To directly compare the signaling capabilities of ERα and GPR30, we expressed each receptor in COS7 cells and examined the signaling pathways contributing to estrogen-mediated mobilization of intracellular calcium and the activation of PI3K (Revankar et al., 2005
). Our results demonstrated that only in the case of GPR30 was EGFR transactivation required, consistent with Filardo’s original observations (Filardo et al., 2000
). Furthermore, that GPR30 is the sole receptor responsible for mediating PI3K activation in response to estrogen via EGFR transactivation in ER-negative breast cancer cells such as SKBr3 is demonstrated by the loss of estrogen-responsiveness following GPR30 antisense transfection. Here antisense transfection reduces both GPR30 protein expression and fluorescent estrogen binding commensurate with the loss in functional responsiveness. Our results also demonstrate that tamoxifen activates PI3K through GPR30 but not ERα, suggesting a possible involvement in tamoxifen-resistant breast cancers and/or the increased incidence and severity of endometrial cancers in women treated with tamoxifen (Rieck et al., 2005
; Senkus-Konefka et al., 2004
). The circumstances under which ERα and/or GPR30 activation lead to EGFR transactivation remain to be fully determined.
PI3K is traditionally activated at the plasma membrane in response to growth factor receptor or G protein-coupled receptor stimulation, resulting in PIP3 accumulation at the plasma membrane. However, we observed PIP3 accumulation in the nucleus in response to estrogen stimulation of both ERα and GPR30. That this was the result of de novo
synthesis of PIP3 was confirmed by pharmacological inhibition of PI3K activity. Although our understanding of the role of nuclear phosphoinositides is limited (Lian and Di Cristofano, 2005
; Neri et al., 2002
), recent studies confirm the presence of PI3K, PDK1, Akt and Pten in the nucleus (Lian and Di Cristofano, 2005
). Additional work has revealed that the activity of steroidogenic factor-1 and liver receptor homolog 1, two members of the nuclear receptor superfamily of transcription factors thought to exhibit ligand-independent activity, is regulated by binding phosphatidylinositol-3,4,5 trisphoshosphate (Krylova et al., 2005
). Thus, accumulation of nuclear PIP3 in response to estrogen-mediated activation of ERα and/or GPR30 may regulate gene expression through this family of nuclear receptors. Additional functions of PIP3 in the nucleus also remain to be explored.