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Molecular and Cellular Biology, March 2003, p. 1633-1646, Vol. 23, No. 5
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.5.1633-1646.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Identification of a Structural Determinant Necessary for the Localization and Function of Estrogen Receptor {alpha} at the Plasma Membrane

Mahnaz Razandi,1,2 Gordon Alton,3 Ali Pedram,1,2 Sanjiv Ghonshani,1,2 Paul Webb,4 and Ellis R. Levin1,2,5*

Division of Endocrinology, Veterans Affairs Medical Center, Long Beach, Long Beach, California 90822,1 Departments of Medicine,2 Pharmacology, University of California, Irvine, Irvine, California 92717,5 Signal Pharmaceuticals Inc., San Diego, California 92121,3 Diabetes and Metabolic Research Unit at the University of California, San Francisco, San Francisco, California 941434

Received 19 July 2002/ Returned for modification 29 August 2002/ Accepted 6 December 2002

Estrogen receptors (ER) have been localized to the cell plasma membrane (PM), where signal transduction mediates some estradiol (E2) actions. However, the precise structural features of ER that result in membrane localization have not been determined. We obtained a partial tryptic peptide/mass spectrometry analysis of membrane mouse ER{alpha} protein. Based on this, we substituted alanine for the determined serine at amino acid 522 within the E domain of wild-type (wt) ER{alpha}. Upon transfection in CHO cells, the S522A mutant ER{alpha} resulted in a 62% decrease in membrane receptor number and reduced colocalization with caveolin 1 relative to those with expression of wt ER{alpha}. E2 was significantly less effective in stimulating multiple rapid signals from the membranes of CHO cells expressing ER{alpha} S522A than from those of CHO cells expressing wt ER{alpha}. In contrast, nuclear receptor expression and transcriptional function were very similar. The S522A mutant was also 60% less effective than wt ER{alpha} in binding caveolin 1, which facilitates ER transport to the PM. All functions of ER{alpha} mutants with other S-to-A substitutions were comparable to those of wt ER, and deletion of the A/B or C domain had little consequence for membrane localization or function. Transfection of ER{alpha} S522A into breast cancer cells that express native ER downregulated E2 binding at the membrane, signaling to ERK, and G1/S cell cycle events and progression. However, there was no effect on the E2 transactivation of an ERE-luciferase reporter. In summary, serine 522 is necessary for the efficient translocation and function of ER{alpha} at the PM. The S522A mutant also serves as a dominant-negative construct, identifying important functions of E2 that originate from activating PM ER.

* Corresponding author. Mailing address: Medical Service (111-I), Long Beach VA Medical Center, 5901 E. 7th St., Long Beach, CA 90822. Phone: (562) 826-5748. Fax: (562) 826-5515. E-mail: ellis.levin{at}med.va.gov.

Molecular and Cellular Biology, March 2003, p. 1633-1646, Vol. 23, No. 5
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.5.1633-1646.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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