The Human Glucocorticoid Receptor Isoform: EXPRESSION, BIOCHEMICAL PROPERTIES, AND PUTATIVE FUNCTION

Alternative splicing of the human glucocorticoid receptor (hGR) primary transcript produces two receptor isoforms, hGRalpha and hGRbeta, which differ at their carboxyl termini. The hGRalpha isoform conveys endocrine information to target tissues by altering patterns of gene expression in a hormone-dependent fashion. In contrast to hGRalpha, very little is known about the hGRbeta splice variant. Using hGRalpha- and hGRbeta-specific riboprobes on human multiple tissue Northern blots, we show that the hGRbeta message has a widespread tissue distribution. We also prove by reverse transcriptase-polymerase chain reaction that the alternative splicing event underlying the formation of the hGRbeta message occurs in these tissues. Because the hGRbeta protein differs from hGRalpha at the extreme COOH terminus, we investigated several of the biochemical properties of hGRbeta expressed in transfected cells. hGRbeta does not bind the glucocorticoid agonist dexamethasone nor the glucocorticoid antagonist RU38486 in vivo. Moreover, in contrast to hGRalpha, hGRbeta is located primarily in the nucleus of transfected cells independent of hormone administration. Finally, in the absence of hGRalpha, hGRbeta is transcriptionally inactive on a glucocorticoid-responsive enhancer. However, when both isoforms are expressed in the same cell, hGRbeta inhibits the hormone-induced, hGRalpha-mediated stimulation of gene expression. Thus, hGRbeta potentially functions as a dominant negative inhibitor of hGRalpha activity

Steroid requirement for androgen receptor dimerization and DNA binding. Modulation by intramolecular interactions between the NH2-terminal and steroid-binding domains.

Infection of Spodoptera frugiperda Sf9 insect cells with recombinant human androgen receptor (AR) baculovirus results in expression of a 118-kDa phosphoprotein that displays high affinity androgen binding and androgen-dependent targeting to the nucleus. Using the DNA mobility shift assay, specific in vitro binding of full-length AR to androgen response element DNA (ARE) requires intracellular hormone exposure. The ability of a variety of steroids to induce ARE binding paralleled their transcriptional potential. Certain antihormones, cyproterone acetate and RU486, promote ARE binding, but a pure antiandrogen, hydroxyflutamide, inhibits AR binding to ARE DNA. AR dimerization requires incubation of recombinant baculovirus-infected insect cells with androgen, but only when one or both components of the dimer contain the NH2-terminal domain. Based on the intensities of ARE binding and lack of binding to an ARE half-site, it appears that, unlike the glucocorticoid receptor, AR binds DNA primarily as a dimer. Thus, full-length baculovirus-expressed AR requires intracellular hormone exposure for dimerization and ARE binding to overcome inhibition imposed by the AR NH2-terminal domain. Antihormones with agonist activity promote dimerization and ARE binding, while a pure antiandrogen blocks AR DNA binding. It is concluded that intramolecular interactions between the NH2-terminal and steroid-binding domains are regulated by the specificity of hormone binding and modulate receptor dimerization and DNA binding

A ligand-dependent bipartite nuclear targeting signal in the human androgen receptor. Requirement for the DNA-binding domain and modulation by NH2-terminal and carboxyl-terminal sequences.

The amino acid sequence requirements for androgen-dependent androgen receptor nuclear import were determined by immunostaining transiently expressed full-length wild type and mutant human androgen receptors (AR) in monkey kidney COS cells and measuring transcriptional activity by cotransfection with a luciferase reporter vector in monkey kidney CV1 cells. Mutagenesis studies revealed a bipartite nuclear targeting sequence in the DNA binding and hinge regions at amino acids 617-633, consisting of two clusters of basic amino acids separated by 10 amino acids, (sequence: see text). In a series of deletion mutants, AR NH2-terminal fragments (residues 1-639 through 1-723) displayed constitutive nuclear import, and transcriptional activity was similar to that of the ligand-activated full-length wild type AR. In contrast, nuclear import and transcriptional activation were inhibited by sequence extensions into the steroid-binding domain (1-771). Constitutive nuclear import was regained in part by NH2-terminal deletions of full-length AR. Expression of AR/pyruvate kinase chimeras defined a sequence required for pre-dominant nuclear localization as residues 580-661, comprised of the second zinc finger region of the DNA-binding domain, the 17-amino-acid putative targeting sequence, and 28 residues of flanking carboxyl-terminal sequence. These studies suggest that the bipartite nuclear targeting sequence of AR includes flanking sequence and is modulated by interactions between the NH2-and carboxyl-terminal regions

Histochemical approaches for the localization of steroid hormone receptors.

Histochemical identification and visualization of steroid hormone receptors remains a goal, despite earlier success with dry- and thaw-mount autoradiography. Applications of various histochemical and immunohistochemical procedures for the identification of steroid hormone target cells and subcellular binding sites are reviewed. Results obtained with dry- and thawmount autoradiography after administration of [ H] estradiol are contrasted with those obtained by liquid emulsion autoradiography, incubation with conjugated estradiol, antibodies to estradiol, or antibodies to estradiol “receptor”. Dry- and thaw-mount autoradiography after single injection of [ H] estradiol, and after tissue slice incubation with [ H] estradiol consistently show preferential nuclear concentration of radioactivity, little in cytoplasm, and none in nucleoli. In contrast, liquid emulsion autoradiography after uterine section incubation with [ H] estradiol shows no nuclear uptake, but only labeling of cytoplasm of eosinophils. Eosinophils are also labeled with conjugated estradiol. Histochemical studies with conjugated estradiol or estradiol antibodies show variable results with preferential cytoplasmic and distinct nucleolar labeling of uterine epithelial and stromal cells; however, there is only occasional nuclear labeling, and some lack of identification of target cells, when compared to results obtained with our autoradiographic techniques. The specificity and utility of certain histochemical techniques need to be established, since some of the results are probably related to technique and do not reflect biological events. Development and use of refined histochemical techniques are needed to further contribute to the clarification of the mechanism of steroid hormone action and to provide a tool for the clinical diagnosis of steroid hormone dependent tumors