ER-X: a novel, plasma membrane-associated, putative estrogen receptor that is regulated during development and after ischemic brain injury - PubMed (original) (raw)
ER-X: a novel, plasma membrane-associated, putative estrogen receptor that is regulated during development and after ischemic brain injury
C Dominique Toran-Allerand et al. J Neurosci. 2002.
Abstract
We showed previously in neocortical explants, derived from developing wild-type and estrogen receptor (ER)-alpha gene-disrupted (ERKO) mice, that both 17alpha- and 17beta-estradiol elicit the rapid and sustained phosphorylation and activation of the mitogen-activated protein kinase (MAPK) isoforms, the extracellular signal-regulated kinases ERK1 and ERK2. We proposed that the ER mediating activation of the MAPK cascade, a signaling pathway important for cell division, neuronal differentiation, and neuronal survival in the developing brain, is neither ER-alpha nor ER-beta but a novel, plasma membrane-associated, putative ER with unique properties. The data presented here provide further evidence that points strongly to the existence of a high-affinity, saturable, 3H-estradiol binding site (K(d), approximately 1.6 nm) in the plasma membrane. Unlike neocortical ER-alpha, which is intranuclear and developmentally regulated, and neocortical ER-beta, which is intranuclear and expressed throughout life, this functional, plasma membrane-associated ER, which we have designated "ER-X," is enriched in caveolar-like microdomains (CLMs) of postnatal, but not adult, wild-type and ERKO neocortical and uterine plasma membranes. We show further that ER-X is functionally distinct from ER-alpha and ER-beta, and that, like ER-alpha, it is re-expressed in the adult brain, after ischemic stroke injury. We also confirmed in a cell-free system that ER-alpha is an inhibitory regulator of ERK activation, as we showed previously in neocortical cultures. Association with CLM complexes positions ER-X uniquely to interact rapidly with kinases of the MAPK cascade and other signaling pathways, providing a novel mechanism for mediation of the influences of estrogen on neuronal differentiation, survival, and plasticity.
Figures
Fig. 1.
ER-X is neither ER-α nor ER-β.a, Western immunoblots of P7 wild-type and ERKO neocortex and adult wild-type mouse ovary, using antibodies to the LBDs of ER-α (Santa Cruz Biotechnology; MC-20; ovary and neocortex) and ER-β (Zymed; ovary). The apparent MW of ER-X (∼62–63 kDa) is clearly different from the MW of the mouse ER-α (67 kDa) and ER-β (60 kDa) ovarian controls. b, Whereas P7 wild-type neocortex contained both the 67 kDa ER-α and the ∼62–63 kDa ER-X bands, P7 ERKO tissues expressed only the ∼62–63 kDa ER-X band. P7 wild-type and ERKO neocortical CLM preparations were greatly enriched with the ∼62–63 kDa protein. A striking reversal of the ER-α/ER-X ratio was seen in wild-type CLM preparations, in which the ∼62–63 kDa form was highly enriched, whereas authentic 67 kDa ER-α was considerably diminished. c, Absence of ER-β from the plasma membrane, CLM, and non-CLM regions. Note the total absence of ER-β from the wild-type plasma membrane and the CLM and non-CLM fractions. Note also the nuclear concentration of the 60 and 64 kDa isoforms of ER-β. PM, Plasma membrane;non-CLM, non-caveolar-like membrane; CLM, caveolar-like membrane.
Fig. 2.
Characterization and purity of the CLM preparations. a, Western immunoblots of CLMs show enrichment in flotillin, the neuron-specific, integral CLM protein. The purity of CLM preparations was verified by the presence of caveolar-enriched resident proteins such as PKC-α (b), and by the absence of the cytosolic protein paxillin, a cytoskeletal component associated with non-CLM regions (c).
Fig. 3.
ER-X is exquisitely sensitive to picomolar concentrations of 17α- and 17β-estradiol. Western immunoblot of ERK1/2 phosphorylation elicited in wild-type neocortical explants by 17β-estradiol (a) and 17α-estradiol (b). Bottom blots, Reprobing with antibodies to total nonphosphorylated ERK1/2 to verify equal loading of ERK1/2 protein across lanes. pERK, phosphoERK. Densitometry confirmed equal loading. Note that significantly higher levels of 17β-estradiol were required for ERK activation, perhaps reflecting the need in wild-type cultures to overcome the inhibitory effect of ER-α on ERK phosphorylation, which, unlike 17α-estradiol, 17β-estradiol activates as well.
Fig. 4.
Estrogen-induced activation of ERK1/2 in CLMs and PNS. Western immunoblots: a, exposure of highly purified, P7 ERKO neocortical CLMs to 17α-estradiol (0.1 n
m
) and 17β-estradiol (10 n
m
) for 30 min elicited MEK-dependent (U0126) phosphorylation of ERK1 and ERK2. Non-CLM regions were unresponsive. Densitometry confirmed equal loading of protein. b, Exposure of P7 wild-type neocortical PNS to 17α-estradiol (0.1 n
m
) and 17β-estradiol (10 n
m
) for 10 min, 37°C elicited MEK-dependent (U0126) phosphorylation of ERK1 and ERK2. Note that, not only did the ER-α-selective ligand PPT reduce ERK phosphorylation levels below baseline (0) very significantly, but that the level of ERK1/2 phosphorylation, elicited by 17β-estradiol, was also significantly lower than after exposure to 17α-estradiol. This difference may be attributed to the fact that P7 wild-type neocortex is also enriched in ER-α which, because it is activated by 17β- (but not 17-α) estradiol and exerts its inhibitory effect on ERK1/2, as was also seen after exposure to PPT. _Bottom blots,_Reprobing with antibodies to nonphosphorylated ERK1/2 to verify equal loading of ERK protein across lanes. Densitometry confirmed equal loading. c, Densitometric analysis of ERK activation in wild-type PNS shown in b. These findings confirm that ER-α is a strong inhibitor of ERK activation, a measure of which is shown by the ability of PPT to effectively prevent ERK activation even in the face of the strong activation of ERK elicited by the PPT vehicle ethanol.
Fig. 5.
Disruption of cholesterol in CLMs impairs ERK activation. Selective disruption of membrane cholesterol by Nystatin in 9-d-old wild-type neocortical explants decreased the ability of estradiol and the BDNF control to elicit ERK phosphorylation.Bottom blots, Reprobing with antibodies to nonphosphorylated ERK1/2 to verify equal loading of ERK protein across lanes. Densitometry confirmed equal loading.
Fig. 6.
ER-X has homology with the LBD of ER-α. Whole-mount of a P2 ERKO neocortical explant, 17 d in vitro. The culture was stained for ER-α mRNA by in situ hybridization with a 48 base oligonucleotide probe to an α-specific region of the ER-α LBD (BER2; Miranda and Toran-Allerand, 1992) and shows the ER-α-like mRNA hybridization signal in neocortical neurons. Residual, untranslated ER-α mRNA? A splice variant of ER-α mRNA? Or the mRNA for a novel ER, ER-X?
Fig. 7.
Direct evidence in ERKO that ER-X is a neuronal plasma membrane-associated receptor with some homology to the ER-α LBD. A, Using antibodies highly specific for an α-specific region of the LBD of ER-α (C1355), large numbers of immature immunoreactive neocortical ERKO neurons with unstained nuclei are seen. B, The immunoreactivity is clearly localized to the cell membrane and cytoplasm and not in the nucleus.D, E, Antibodies, raised against the full-length ER-α molecule, said to recognize epitopes in the 5′, N-terminal region (6F11), but which we have found also to cross-react significantly with ER-β, show widespread nuclear labeling with no cytoplasmic or membrane labeling seen. The nuclear labeling observed most likely reflects intranuclear ER-β, which is normally expressed in both wild-type and ERKO neocortical neurons. C, CLM association of ER-X in ERKO neocortical neurons was further documented at the ultrastructural level by demonstrating immunoreactive flotillin (gold particles), colocalized with immunoreactivity for the ER-α LBD**(**horseradish peroxidase) on a mushroom-like neocortical dendritic spine. Scale bars, 10 μm.
Fig. 8.
Binding of 3H-estradiol to Percoll-purified plasma membranes from P7 ERKO and wild-type mouse neocortex. A, Identical amounts of membrane protein (50 μg/tube) were incubated with varying concentrations of3H-estradiol (0.3–8 n
m
) for 18 hr at 4C. The reaction was terminated by addition of hydroxylapatite (HAP). The membranes and HAP were sedimented by centrifugation in a microfuge, and the pellet was washed four times to remove free steroid. Radioactivity in the pellets was extracted with ethanol and counted. Nonsaturable binding, assessed in the presence of 1 μ
m
unlabeled DES, was subtracted from the total counts, and the saturable binding was plotted as the ratio of bound–unbound ligand versus the concentration of bound 3H-estradiol. Similar concentrations of high-affinity binding (equilibrium dissociation constant, _K_d, ∼1.6 n
m
) were observed in wild-type and ERKO membranes.B, Specificity of the binding site in Percoll-purified membranes from P7 ERKO mouse neocortex. Aliquots of plasma membrane were incubated with 2 n
m
3H-estradiol for 18 hr at 4°C in the presence and absence of different concentrations (50 n
m
and 1 μ
m
) of 17α-estradiol, 17β-estradiol, or progesterone. Bound 3H-estradiol was separated by sedimentation with HAP and counted at an efficiency of 50%. Data represent the number of bound counts (after subtraction of HAP-only blank control tubes, containing no membrane protein) expressed as the means ± SD of triplicate determinations. The_horizontal dashed line_ indicates the level of nonspecific binding observed in the presence of 1 μ
m
DES.
Fig. 9.
ER-X is developmentally regulated. ER-X expression is developmentally regulated and is maximally expressed at ∼P7–P10 in the neocortex (a) and the uterus (b). During the first postnatal month, wild-type and ERKO neocortical ER-X levels decline dramatically and become barely visible in the adult.
Fig. 10.
ER-X is upregulated after ischemic brain injury in the adult. Comparison of ER-α and ER-X expression in the infarcted and noninfarcted adult neocortex. After a large ischemic infarct in the neocortex produced by middle cerebral artery occlusion, there was not only upregulation of ER-α expression in the penumbra of the ligated, ischemic side but also upregulation of ER-X therein as well, suggesting re-expression of a developmental mechanism normally latent in the adult. Note the lack of significant ER-X expression on the noninfarcted side. MCF-7 mammary tumor cells and adult uterus = ER-α controls; P7 neocortex = ER-X control.
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