Cell Membrane and Nuclear Estrogen Receptors (ERs) Originate from a Single Transcript: Studies of ERα and ERβ Expressed in Chinese Hamster Ovary Cells

Abstract

The existence of a putative membrane estrogen receptor (ER) has been supported by studies accomplished over the past 20 yr. However, the origin and functions of this receptor are not well defined. To study the membrane receptor, we transiently transfected cDNAs for ERalpha or ERbeta into Chinese hamster ovary (CHO) cells. Transfection of ERalpha resulted in a single transcript by Northern blot, specific binding of labeled 17beta-estradiol (E2), and expression of ER in both nuclear and membrane cell fractions. Competitive binding studies in both compartments revealed near identical dissociation constants (K(d)S) of 0.283 and 0.287 nM, respectively, but the membrane receptor number was only 3% as great as the nuclear receptor density. Transfection of ERbeta3 also yielded a single transcript and nuclear and membrane receptors with respective Kd values of 1.23 and 1.14 nM; the membrane receptor number was only 2% compared with expressed nuclear receptors. Estradiol binding to CHO-ERalpha or CHO-ERbeta activated Galphaq and G(alpha)s proteins in the membrane and rapidly stimulated corresponding inositol phosphate production and adenylate cyclase activity. Binding by 17-beta-E2 to either expressed receptor comparably enhanced the nuclear incorporation of thymidine, critically dependent upon the activation of the mitogen-activated protein kinase, ERK (extracellular regulated kinase). In contrast, c-Jun N-terminal kinase activity was stimulated by 17-beta-E2 in ERbeta-expressing CHO, but was inhibited in CHO-ERalpha cells. In summary, membrane and nuclear ER can be derived from a single transcript and have near-identical affinities for 17-beta-E2, but there are considerably more nuclear than membrane receptors. This is also the first report that cells can express a membrane ERbeta. Both membrane ERs activate G proteins, ERK, and cell proliferation, but there is novel differential regulation of c-Jun kinase activity by ERbeta and ERalpha.

Functional significance of the rapid regulation of brain estrogen action: Where do the estrogens come from?

“…1), including the modulation of intracellular calcium [44,55,122,165,175,209] and protein kinase C (PKC; [266,276]). There is also evidence that membrane effects of estrogens can activate intracellular signaling pathways involving cyclic AMP (cAMP; [103,170,254]), protein kinase A (PKA; [104,141,266]), the "mitogen activated protein kinases" (or MAP kinases; [39,122,153,205,260,269,276]), and the tyrosine kinases [39]. The activation of these intracellular signaling pathways results primarily in phosphorylations/dephosphorylations producing different kinds of physiological responses such as the decoupling of a receptor from its effector system [141,[171][172][173] or the modulation of the catalytic activity of an enzyme [191].…”

Membrane-initiated estrogen signaling in hypothalamic neurons

“…However, it is now widely accepted that some of the actions of estrogen are quite rapid and cannot be attributed to the classical nuclear-initiated steroid signaling of ERα or ERβ. One view is that both nuclear and plasma membrane-associated ERs might be products of the same genes (Razandi et al, 1999, Boulware et al, 2005, Pedram et al, 2006, Szegõ et al, 2006, Dewing et al, 2007. This belief stems primarily from the fact that many of the rapid effects of E2 can be induced by selective ERα or ERβ ligands, antagonized by the ER antagonist, ICI 182,780, or are lost in animals bearing mutations in ERα and/or ERβ genes (Couse and Korach, 1999, Singer et al, 1999, Dubal et al, 2001, Wade et al, 2001, Abraham et al, 2003, Boulware et al, 2005.…”

Modulation of hypothalamic neuronal activity through a novel G-protein-coupled estrogen membrane receptor

“…The membrane-associated ERs might be derived from the same genes as ERα and ERβ [45][46][47][48]. This belief stems primarily from the fact that many of the rapid effects of E 2 can be induced by selective ERα or ERβ ligands, antagonized by the ER antagonist, ICI 182,780 or are lost in animals bearing mutations in ERα and/or ERβ genes [15,46,[49][50][51][52].…”