Perimembrane localization of the estrogen receptor alpha protein in neuronal processes of cultured hippocampal neurons.
There is clear evidence of rapid, nongenomic responses to estrogen in a variety of neuronal model systems. To address the question of whether some of these rapid estrogen signals might be transduced by the classical estrogen receptor (ER) alpha or a closely related protein in nontransformed neurons, we undertook the present study using isolated fetal rat hippocampal neurons. Several antibodies developed to detect ERalpha were tested in this system and showed positive membrane staining in nonpermeabilized neurons. MC-20, an affinity purified anti-ERalpha, rabbit polyclonal IgG antibody which does not recognize ERbeta was selected to carry out the majority of the experiments. When permeabilized, the hippocampal neurons exhibited low levels of nuclear staining for ERalpha, but abundant labeling for ERalpha throughout the entire cell including the neurites. In addition to traditional immunocytochemistry controls, incubation of neurons for 24 h in the presence of 10 microM antisense oligonucleotide directed against the translation start site of ERalpha reduced ERalpha immunoreactivity throughout the neurons providing further evidence that the immunostaining was specific for ERalpha. Confocal and conventional microscopy demonstrated that the antigen was predominately extranuclear and localization of ERalpha in the neurites suggests that the receptor is in close proximity to the plasma membrane. This localization is consistent with a role for ERalpha as a transducer of rapid, nongenomic estrogen responses in hippocampal neurons.
Clarke
C H
CH
Department of Pharmacology, University of Texas Medical Branch, Galveston, Tex., USA.
Norfleet
A M
AM
Clarke
M S
MS
Watson
C S
CS
Cunningham
K A
KA
Thomas
M L
ML
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry*; D002478:Cells, Cultured; D047628:Estrogen Receptor alpha; D005260:Female; D005333:Fetus / Q000166:cytology; D005455:Fluorescent Antibody Technique; D006624:Hippocampus / Q000166:cytology*; D008565:Membrane Proteins / Q000032:analysis; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009474:Neurons / Q000737:chemistry* / Q000648:ultrastructure; D011247:Pregnancy; D011817:Rabbits; D051381:Rats; D011960:Receptors, Estrogen / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D014599:Uterus / Q000737:chemistry
D047628:Estrogen Receptor alpha; D008565:Membrane Proteins; D011960:Receptors, Estrogen