Analysis of estrogen receptor alpha signaling complex at the plasma membrane.

There is accumulating evidence that the estrogen receptor (ER) can transduce specific signals at the plasma membrane. We tried to clarify the biological function of ER as a signaling molecule by identifying proteins that interact with the membrane-localized ER. The activation function 1 and 2 (AF-1 and AF-2) domains of ERalpha with or without the membrane-targeting sequence were stably expressed in the breast cancer cell line, MCF-7. The level of tyrosine phosphorylation of AF-2 was significantly elevated by the membrane localization. By mass-spectrometry analysis, alpha- and beta-tubulins and heat shock protein 70 were identified as the AF-1-associated proteins. Of these, tubulins are associated only with membrane-targeted AF-1.