Elsevier

Steroids

Volume 67, Issue 6, May 2002, Pages 429-437
Steroids

The dynamic and elusive membrane estrogen receptor-α

https://doi.org/10.1016/S0039-128X(01)00172-6Get rights and content

Abstract

Many studies have demonstrated the nuclear forms of steroid receptors and their activities, while fewer investigators have identified and described the membrane forms of these receptors. Our immuno-identification approaches for the qualitative and quantitative comparison of the membrane form of the estrogen receptor-α (mERα) to its nuclear counterpart now allow us to address questions about the comparative levels and regulation of these receptor forms. ERα-specific antisense oligonucleotides eliminate mERα expression, while only mildly reducing the nuclear ERα. Success of immuno-identification for the mERα is very sensitive to different fixation protocols, affecting cell permeability (and thus distinction from the intracellular form) and differential epitope preservation. All such identifications must be accompanied by proof of cell membrane integrity and focal plane assessments. The mERα expression on selected cells declines rapidly with cell passage number and cell density. Expression of mERα is enhanced by serum starvation and selection for specific phases of the cell cycle. The hinge region of the protein is sensitive to ligand-induced epitope masking and to antibody-induced changes in receptor-mediated responses. Responsive cells are often diluted within cell populations by loss of the membrane receptor form. The bimodality of the rapid estrogen action, with inhibitory doses between picomolar and nanomolar stimulatory concentrations, requires detailed dose-response curves. Finally, responsive cells can be lost from assays, as upon estrogen treatment they rapidly round up and leave the substrates to which they are attached. These regulatory phenomena demonstrate that levels of the membrane form of the estrogen receptor are very dynamic.

Introduction

We have previously used the properties of multiple antibodies (Abs) to identify the membrane form of estrogen receptor-α (mERα) (summarized in Fig. 1) and probe its membrane orientation and function. More recently, we have developed Ab-based techniques and experimental designs to quantify different subcellularly located forms of estrogen receptor-α (membrane and nuclear). We have focused our studies on the expression of mERα in cells in which they are naturally expressed, unaided by transfection of ERα cDNAs into recipient receptorless cells.

The ability to separate populations of cells into those enriched and depleted for membrane steroid receptor expression [1], [2], [3], [4] has been essential to demonstrating affiliated functions, because only a subset of cells in clonal populations have mERα. Once selected, the drift of clonal cell populations back to mERα-negative status constantly threatens studies designed to demonstrate functional correlates. This intriguing transitory presence of mERα caused us to review our past data for clues to mERα regulation and provided guidelines for specific study of the regulatory influences on mERα in order to better control our functional studies. As we now use these measurement tools to ask questions about the comparative levels and regulatory influences on membrane vs. intracellular estrogen receptors, we may discover more about the cooperative nature of these forms and their separate functions.

Section snippets

Cell culture

GH3/B6/F10 (F10) cells were subcloned from GH3/B6 cells (a gift from Dr. Bernard Dufy) by limiting dilution cloning, as previously described [1]. These cells were routinely cultured at 37°C in serum-supplemented medium consisting of Ham’s F10 medium (GIBCO-BRL, Grand Island, NY), 12.5% heat-inactivated horse serum, 2.5% heat-inactivated defined-supplemented calf serum, and 1.5% heat-inactivated fetal calf serum (FCS) (all sera supplied by Hyclone, Logan, UT). In preparation for these

The influence of fixation method on cell permeability and resulting distinctions between receptor forms

Cell types vary tremendously in the lipid and protein composition of their membranes, and so are variably penetrated by detergents and the components of various fixative mixtures. In working with a protein which is present both in the membrane and in the nucleus, careful attention to the ingredients and concentrations of fixatives, and the length of treatment times is necessary to delineate receptor populations in specific subcellular locations. In the case of ERα, this is particularly

Acknowledgments

C.H.C. was supported by the Zelda Zinn Casper Fellowship Fund, the NIEHS Toxicology Training Grant 5232ES07254, and the UTMB Centennial Center for Environmental Toxicology. The authors wish to thank Dr. Darrell Carney for use of his plate reader and microscope and Dr. David Konkel for critical reading of the manuscript. Both are from the Univ. of Texas Medical Branch, Galveston, TX.

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