In 2000, Filardo and colleagues demonstrated that the rapid activation of ERK in breast cancer cells was dependent upon the presence of GPR30/GPER (Filardo et al., 2000
). Exogenous expression of GPER in MDA-MB-231 breast cancer cells, which express little to no ERα or GPER, resulted in estrogen-dependent ERK1/2 phosphorylation, similar to the response in MCF-7 cells, which express both ERα and GPER. Estrogen-dependent ERK1/2 phosphorylation was also observed in SKBr3 breast cancer cells, which express GPER but lack ERα. Together, these results indicated that GPER expression was critical for the rapid activation of ERK.
Studies by other groups in the following years demonstrated that GPER was involved in the upregulation of nerve growth factor in macrophages (Kanda and Watanabe, 2003a
), and cyclin D2 and Bcl-2 in keratinocytes (Kanda and Watanabe, 2003b
; Kanda and Watanabe, 2004
). In addition, estrogen-mediated activation of c-fos
transcription in breast cancer cells was also shown to occur in a GPER-dependent manner (Maggiolini et al., 2004
). In these studies the requirement for GPER was established via knockdown of GPER in cells that endogenously expressed the receptor. Such observations indicated that although classical estrogen receptors were traditionally associated with transcriptional regulation, GPER was also capable of mediating estrogen-dependent gene expression.
Despite these correlative demonstrations, the mechanistic link between GPER expression and estrogen signaling remained ambiguous due in large part to a history of similar rapid signaling events being ascribed to ERα(Razandi et al., 2003
). In an effort to resolve this controversy, two studies in 2005 described the binding of estrogen to GPER-expressing cells. Thomas et al
. reported the specific binding of tritiated estrogen to membranes of SKBr3 (ERα- and ERβ-negative, GPER-positive) breast cancer cells and to GPER-transfected human embryonic kidney (HEK) cells (Thomas et al., 2005
). The reported binding constant of 3 nM was approximately 10-fold poorer than the value typically reported for estrogen binding to the ligand-binding domain of ERα (Kuiper et al., 1997
). In addition, estrogen binding was absent in untransfected HEK cells and reduced in SKBr3 cells treated with siRNA targeting GPER.
In contrast to tritiated estrogen binding, Revankar et al
. visualized the cellular and subcellular binding of estrogen to GPER employing a novel fluorescent estrogen derivative (Revankar et al., 2005
). Confocal microscopy demonstrated that binding to GPER occurred in the endoplasmic reticulum with no detectable binding at the plasma membrane, consistent with antibody staining of the endogenously expressed receptor in numerous cell types. This subcellular pattern of localization has been confirmed in a number of subsequent studies (Brailoiu et al., 2007
; Matsuda et al., 2008
; Otto et al., 2008b
; Sakamoto et al., 2007
), as well as through the use of selectively membrane-permeable estrogen derivatives (Revankar et al., 2007
), although reports to the contrary also exist (Funakoshi et al., 2006
; Thomas et al., 2005
). Increasing evidence supports the intracellular localization and function of certain GPCRs, including lipid-activated GPCRs (for prostaglandins, platelet-activating factor and lysophosphatidic acid), and associated signaling molecules including G proteins and ion channels (Zhu et al., 2006
Flow cytometric analysis of GPER-transfected cells by Revankar et al.
demonstrated that estrogen binding was proportional to GPER expression, similar to that observed in cells transfected with either ERα or ERβ, supporting the conclusion that GPER expression directly generates estrogen-binding sites (Revankar et al., 2005
). Competition binding assays revealed a binding constant for estrogen of approximately 6 nM, similar to the value of 3 nM reported by Thomas et al.
using a completely different approach (Thomas et al., 2005
). These two studies strongly implicated GPER as an estrogen-binding protein with low nM affinity, alleviating one of the barriers to the acceptance of GPER as a true estrogen receptor.