Distinctive Actions of Membrane-Targeted Versus Nuclear Localized Estrogen Receptors in Breast Cancer Cells

Abstract

Estrogens regulate multiple activities in breast cancer cells, including proliferation. Whereas these hormones are most commonly known to regulate gene transcription through direct interaction with estrogen receptors (ERs) and with specific DNA sequences of target genes, recent studies show that ER also activates a number of rapid signaling events that are initiated at the cell membrane. To study the membrane-initiated effects of estrogen and separate them from the activities initiated by the nuclear localized ER in human breast cancer cells, we generated MDA-MB-231 breast cancer cell lines that have stably integrated either the wild-type nuclear form of ER (WT-ER) or a modified, membrane-targeted ER (MT-ER) that lacks a nuclear localization sequence and is dually acylated with a myristoylation sequence at the N terminus and a palmitoylation sequence at the C terminus. We demonstrate that MT-ER is membrane localized in the absence of estradiol (E2), showing punctate membrane and cytoplasmic speckles after E2 exposure. In contrast to WT-ER, MT-ER was not down-regulated by E2 or by antiestrogen ICI 182,780 exposure, and MT-ER failed to regulate endogenous E2-responsive genes highly up-regulated by WT-ER. Cells expressing MT-ER showed a greater serum response element-mediated transcriptional response that was partially inhibited by antiestrogen ICI 182,780. The MT-ER and WT-ER differentially altered ERK1/2 and Akt activities and the proliferation of breast cancer cells in response to E2. Hence, this study reveals distinct actions of the MT-ER vs. the WT-ER in effecting estrogen actions in breast cancer cells.

Phosphoinositide 3-Kinase-independent Non-genomic Signals Transit from the Androgen Receptor to Akt1 in Membrane Raft Microdomains

“…The p/m-AR described in the current study is structurally different from the membrane-targeted ER described by Rai et al (48). Membrane-targeted-ER was engineered by N-terminal myristoylation and C-terminal palmitoylation, while lacking a nuclear localization sequence (48).…”

“…Membrane-targeted-ER was engineered by N-terminal myristoylation and C-terminal palmitoylation, while lacking a nuclear localization sequence (48). In contrast, we engineered p/m-AR to express a short peptide with a myristoylation and palmitoylation site linked to the N-terminal domain of AR.…”

“…Although structurally distinct, both p/m-AR and membranetargeted ER displayed similar characteristics: localization to the plasma membrane and cytoplasm, transcriptional inactivity, and rapid response to their cognate ligands. This suggests the possibility that minimal post-translational modification of the AR may be sufficient to elicit non-genomic effects of androgen (48).…”

Induction of Osteoblast Differentiation by Selective Activation of Kinase-Mediated Actions of the Estrogen Receptor

“…During the last few years, we and others have identified several synthetic compounds that, unlike estradiol, can selectively activate kinase-initiated routes by which the estrogen receptor (ER) controls gene transcription (28,38,60). By use of these compounds, substantial evidence that kinase-mediated effects on gene transcription are dissociable from direct cis or trans effects on transcription has been obtained (28,38,39,54). Moreover, the use of selective activators of kinases, as well as compounds that dissociate the classical genomic actions of the estrogen receptor from cross talk with other transcription factors (8,56), has provided extensive proof of the general concept that it is possible to eliminate the uterotropic activity of estrogens while retaining other nonreproductive actions.…”

17β-Estradiol and tamoxifen stimulate rapid and transient ERK activation in MCF-7 cells via distinct signaling mechanisms

“…[18][19][20] This can be partially explained by differences in the expression levels of ER, EGFR, and the HER-2 receptor. [21][22][23][24] Furthermore, it has been suggested that multiple mechanisms exist by which estrogen stimulation can lead to ERK activation. 15,25 This underscores the need for a study using an appropriate cell type and consistent experimental conditions to elucidate the mechanisms by which E2 and tamoxifen induce ERK phosphorylation.…”