Intracellular gonadotropin-releasing hormone receptors in breast cancer and gonadotrope lineage cells

Abstract

Gonadotropin-releasing hormone receptors (GnRHRs) are expressed in gonadotropes and several extra-pituitary sites. They are assumed to be cell surface proteins but the human (h) GnRHR lacks features favoring plasma membrane localization and receptor location varies with cell type. When expressed in mammary (MCF7) cells, cell surface hGnRHR binding was much lower than that of mouse and sheep GnRHRs (type I GnRHRs without C-terminal tails), Xenopus (X) and marmoset type II GnRHRs (type II GnRHRs with C-tails) or chimeric receptors (type I GnRHRs with added XGnRHR C-tails). hGnRHR binding was higher in aT4 (gonadotrope-derived) cells and was increased less by C-tail addition. Whole cell levels of tagged human, Xenopus and chimeric GnRHRs were comparable (Western blotting) and confocal microscopy revealed that the hGnRHR is primarily intracellular (distribution similar to the endoplasmic reticulum marker, calreticulin), whereas most XGnRHR is at the plasma membrane, and adding the C-tail increased cell surface hGnRHR levels. A membrane-permeant antagonist increased cell surface hGnRHR number (O4-fold, t 1 ⁄ 2 Z 4 h) and also increased hGnRHR signaling and hGnRHRmediated inhibition of proliferation. A more rapid increase in hGnRHR binding occurred when the temperature was raised from 4 to 37 8C (O5-fold, t 1 ⁄ 2 Z15 min) and this effect was prevented by mutation to prevent signaling. Thus, cell surface GnRHR expression depends on receptor and cell type and the hGnRHR is primarily an intracellular protein that traffics to the cell surface for signaling in MCF7 cells. Manipulations favoring such trafficking may facilitate selective targeting of extra-pituitary GnRHRs.

The Human Gonadotropin Releasing Hormone Type I Receptor Is a Functional Intracellular GPCR Expressed on the Nuclear Membrane

“…Our spatial observations are consistent with those previously reported by Sedgley et al [26] who also observed that in mammary (MCF7) cells the wild-type human GnRH-RI is inefficiently expressed at the plasma membrane. Our study is also consistent with previous biochemical data [8], [11], [27]–[31] that suggest both the wild-type and naturally occurring mutant human GnRH-RI are expressed inefficiently at the plasma membrane and are retained intracellularly, both in the presence and absence of agonist.…”

“…It also seems that in HEK 293 cells, Xenopus cytoplasmic tail sequences are not sufficient to visibly increase plasma membrane expression of the hGnRH-RI. This observation was unexpected since studies have demonstrated that cytoplasmic tail sequences potentiate the plasma membrane expression of GPCRs [50], [51], including that of the human GnRH-RI [26]. The differences observed in our HEK 293 cell-based study compared to the MCF7 cell-based study by Sedgley et al [26] suggests that nuclear localization might be cell type-dependent.…”

“…This observation was unexpected since studies have demonstrated that cytoplasmic tail sequences potentiate the plasma membrane expression of GPCRs [50], [51], including that of the human GnRH-RI [26]. The differences observed in our HEK 293 cell-based study compared to the MCF7 cell-based study by Sedgley et al [26] suggests that nuclear localization might be cell type-dependent. This conclusion is in full agreement with those of Finch et al [52] who show variations in surface expression of the human GnRH-RI and chimeric HX-GnRH-RI among various cell lines reported to endogenously express GnRH-RI.…”

Human gonadotropin-releasing hormone receptor-activated cellular functions and signaling pathways in extra-pituitary tissues and cancer cells (Review)

“…2D). In addition, cellular proliferation decreased after addition of Buserelin or other GnRH agonists, this depended either on the amount of GnRH at cell surfaces or on receptor functionality (61,124). Concerning the molecular mechanism employed by GnRHR in breast cancer cells to reduce cell proliferation in MCF-7 cells, the direct inhibitory effects of Buserelin on breast cancer cells was mediated, at least in part, by an antagonists effect on the EGF receptor (125) and IGF receptor (126) (Fig.…”

Gonadotropin-releasing hormone neuropeptides and receptor in human breast cancer: Correlation to poor prognosis parameters

“…Growth effects of GnRH analogs on breast cancer cells depend on the amount of GnRH at cell surfaces and on receptor functionality [12,31], therefore quantification of expression levels holds a specific interest to predict cellular responses. Furthermore, GnRH receptors may contribute to the breast tumor responsiveness to pharmacotherapy with GnRH analogs and thus estimation of expression levels could define a more sensitive patient subpopulation.…”