This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cidlowski, J. A. Articles by Sar, M. Search for Related Content PubMed PubMed Citation Articles by Cidlowski, J. A. Articles by Sar, M.

Novel Antipeptide Antibodies to the Human Glucocorticoid Receptor: Recognition of Multiple Receptor Forms in Vitro and Distinct Localization of Cytoplasmic and Nuclear Receptors

Department of Physiology, Lineberger Cancer Center Cell Biology Program, University of North Carolina Chapel Hill, North Carolina 27599
Department of Biochemistry, Lineberger Cancer Center Cell Biology Program, University of North Carolina Chapel Hill, North Carolina 27599
Department of Pathology, Lineberger Cancer Center Cell Biology Program, University of North Carolina Chapel Hill, North Carolina 27599
Department of Pediatrics, Lineberger Cancer Center Cell Biology Program, University of North Carolina Chapel Hill, North Carolina 27599
Department of Cell Biology and Anatomy, Lineberger Cancer Center Cell Biology Program, University of North Carolina Chapel Hill, North Carolina 27599

Address requests for reprints to: John A. Cidlowski, 460 Medical Sciences Research Building, Department of Physiology, CB 7545, University of North Carolina, Chapel Hill, North Carolina 27599.

Abstract

We have synthesized two peptides that correspond to unique regions of the amino-terminus of the human glucocorticoid receptor (GR). Peptides representing amino acids 245–259 and 346–367 (designated 59 and 57, respectively) were chosen on the basis of hydrophobicity/hydrophilicity ratios as well as overall proline content. These peptides were then used as antigens to produce epitope-specific antibodies that recognize and interact with human GR in a variety of physical states. Antiserum directed against each peptide recognizes denatured, [³H] dexamethasone mesylate-labeled GR as well as unliganded receptor on Western blots. In contrast to other antipeptide GR antibodies, these antibodies recognize and form stable complexes with unactivated and molybdate-stabilized forms of the GR, indicating that neither epitope is occluded when the receptor exists in an oligomeric state. Activated, 4S DNA-binding forms of the receptor are also recognized by both antibodies. The interaction of antibodies 59 and 57 with human GR in various states is highly specific based on the observation that preincubation of either antiserum with the appropriate peptide completely precludes the recognition of receptor by antibody. Titration analysis of antisera reveals that an increase in the antibody concentration causes discrete increases in the sedimentation coefficient of GR on sucrose gradients. These shifts occur under high salt conditions and are consistent with the formation of multiple stable antibody-receptor complexes. Interestingly, neither antibody interferes with the ability of the GR to be activated into a DNA-binding form or with the ability of the activated GR to interact with DNA cellulose. Consistent with these observations, both antibodies recognize and form stable complexes with GR when the receptor is associated with DNA fragments that contain specific glucocorticoid-responsive elements. Thus, both antibodies appear to recognize all known forms of the human GR protein. Using immunohistochemical techniques to visualize GR in HeLa S₃ cells as well as in Chinese hamster ovary cells that stably express transfected human GR, a cytoplasmic location for receptor is observed in the absence of ligand. In contrast, immunoreactive GR, is predominantly nuclear after hormone treatment, further supporting a role for nuclear translocation in GR function.

FOOTNOTES

This work was supported by Grants DK-32459 and DK-32460 from the NIH.

Received for publication May 10, 1990. Revision received June 15, 1990. Accepted for publication June 15, 1990.

This article has been cited by other articles:

				A. J. Galliher-Beckley, J. G. Williams, J. B. Collins, and J. A. Cidlowski Glycogen Synthase Kinase 3{beta}-Mediated Serine Phosphorylation of the Human Glucocorticoid Receptor Redirects Gene Expression Profiles Mol. Cell. Biol., December 15, 2008; 28(24): 7309 - 7322. [Abstract] [Full Text] [PDF]

				N. Z. Lu, J. B. Collins, S. F. Grissom, and J. A. Cidlowski Selective Regulation of Bone Cell Apoptosis by Translational Isoforms of the Glucocorticoid Receptor Mol. Cell. Biol., October 15, 2007; 27(20): 7143 - 7160. [Abstract] [Full Text] [PDF]

				C. M. Jewell and J. A. Cidlowski Molecular Evidence for a Link between the N363S Glucocorticoid Receptor Polymorphism and Altered Gene Expression J. Clin. Endocrinol. Metab., August 1, 2007; 92(8): 3268 - 3277. [Abstract] [Full Text] [PDF]

				N. Z. Lu, S. E. Wardell, K. L. Burnstein, D. Defranco, P. J. Fuller, V. Giguere, R. B. Hochberg, L. McKay, J.-M. Renoir, N. L. Weigel, et al. International Union of Pharmacology. LXV. The Pharmacology and Classification of the Nuclear Receptor Superfamily: Glucocorticoid, Mineralocorticoid, Progesterone, and Androgen Receptors Pharmacol. Rev., December 1, 2006; 58(4): 782 - 797. [Full Text] [PDF]

				H. Russcher, P. Smit, E. L. T. van den Akker, E. F. C. van Rossum, A. O. Brinkmann, F. H. de Jong, S. W. J. Lamberts, and J. W. Koper Two Polymorphisms in the Glucocorticoid Receptor Gene Directly Affect Glucocorticoid-Regulated Gene Expression J. Clin. Endocrinol. Metab., October 1, 2005; 90(10): 5804 - 5810. [Abstract] [Full Text] [PDF]

				B. M. Necela and J. A. Cidlowski A Single Amino Acid Change in the First Zinc Finger of the DNA Binding Domain of the Glucocorticoid Receptor Regulates Differential Promoter Selectivity J. Biol. Chem., September 17, 2004; 279(38): 39279 - 39288. [Abstract] [Full Text] [PDF]

				M. A. Hermoso, T. Matsuguchi, K. Smoak, and J. A. Cidlowski Glucocorticoids and Tumor Necrosis Factor Alpha Cooperatively Regulate Toll-Like Receptor 2 Gene Expression Mol. Cell. Biol., June 1, 2004; 24(11): 4743 - 4756. [Abstract] [Full Text] [PDF]

				M. J. M. Schaaf and J. A. Cidlowski Molecular Determinants of Glucocorticoid Receptor Mobility in Living Cells: the Importance of Ligand Affinity Mol. Cell. Biol., March 15, 2003; 23(6): 1922 - 1934. [Abstract] [Full Text] [PDF]

				L. Dufourny and D. C. Skinner Progesterone Receptor, Estrogen Receptor {alpha}, and the Type II Glucocorticoid Receptor Are Coexpressed in the Same Neurons of the Ovine Preoptic Area and Arcuate Nucleus: A Triple Immunolabeling Study Biol Reprod, November 1, 2002; 67(5): 1605 - 1612. [Abstract] [Full Text] [PDF]

				L. Dufourny and D. C. Skinner Influence of Estradiol on NADPH Diaphorase/Neuronal Nitric Oxide Synthase Activity and Colocalization with Progesterone or Type II Glucocorticoid Receptors in Ovine Hypothalamus Biol Reprod, September 1, 2002; 67(3): 829 - 836. [Abstract] [Full Text] [PDF]

				C. L. Mann, C. D. Bortner, C. M. Jewell, and J. A. Cidlowski Glucocorticoid-Induced Plasma Membrane Depolarization during Thymocyte Apoptosis: Association with Cell Shrinkage and Degradation of the Na+/K+-Adenosine Triphosphatase Endocrinology, December 1, 2001; 142(12): 5059 - 5068. [Abstract] [Full Text] [PDF]

				A. D. Wallace and J. A. Cidlowski Proteasome-mediated Glucocorticoid Receptor Degradation Restricts Transcriptional Signaling by Glucocorticoids J. Biol. Chem., November 9, 2001; 276(46): 42714 - 42721. [Abstract] [Full Text] [PDF]

				C. L. Mann and J. A. Cidlowski Glucocorticoids Regulate Plasma Membrane Potential During Rat Thymocyte Apoptosis in Vivo and in Vitro Endocrinology, January 1, 2001; 142(1): 421 - 429. [Abstract] [Full Text] [PDF]

				L. I. McKay and J. A. Cidlowski CBP (CREB Binding Protein) Integrates NF-{kappa}B (Nuclear Factor-{kappa}B) and Glucocorticoid Receptor Physical Interactions and Antagonism Mol. Endocrinol., August 1, 2000; 14(8): 1222 - 1234. [Abstract] [Full Text]

				New Resistance to Several Steroids in Two Sisters J. Clin. Endocrinol. Metab., December 1, 1999; 84(12): 4454 - 4464. [Abstract] [Full Text]

				R. H. Oakley, C. M. Jewell, M. R. Yudt, D. M. Bofetiado, and J. A. Cidlowski The Dominant Negative Activity of the Human Glucocorticoid Receptor beta Isoform. SPECIFICITY AND MECHANISMS OF ACTION J. Biol. Chem., September 24, 1999; 274(39): 27857 - 27866. [Abstract] [Full Text] [PDF]

				C. M. Pariante, B. D. Pearce, T. L. Pisell, C. I. Sanchez, C. Po, C. Su, and A. H. Miller The Proinflammatory Cytokine, Interleukin-1{alpha}, Reduces Glucocorticoid Receptor Translocation and Function Endocrinology, September 1, 1999; 140(9): 4359 - 4366. [Abstract] [Full Text]

				R. K. Tyagi, L. Amazit, P. Lescop, E. Milgrom, and A. Guiochon-Mantel Mechanisms of Progesterone Receptor Export from Nuclei: Role of Nuclear Localization Signal, Nuclear Export Signal, and Ran Guanosine Triphosphate Mol. Endocrinol., November 1, 1998; 12(11): 1684 - 1695. [Abstract] [Full Text]

				L. I. McKay and J. A. Cidlowski Cross-Talk between Nuclear Factor-{kappa}B and the Steroid Hormone Receptors: Mechanisms of Mutual Antagonism Mol. Endocrinol., January 1, 1998; 12(1): 45 - 56. [Abstract] [Full Text]

				R. H. Oakley, J. C. Webster, M. Sar, C. R. Parker Jr., and J. A. Cidlowski Expression and Subcellular Distribution of the {beta}-Isoform of the Human Glucocorticoid Receptor Endocrinology, November 1, 1997; 138(11): 5028 - 5038. [Abstract] [Full Text] [PDF]

				J. Yang, J. Liu, and D. B. DeFranco Subnuclear Trafficking of Glucocorticoid Receptors In Vitro: Chromatin Recycling and Nuclear Export J. Cell Biol., May 5, 1997; 137(3): 523 - 538. [Abstract] [Full Text] [PDF]

				J. C. Webster, C. M. Jewell, J. E. Bodwell, A. Munck, M. Sar, and J. A. Cidlowski Mouse Glucocorticoid Receptor Phosphorylation Status Influences Multiple Functions of the Receptor Protein J. Biol. Chem., April 4, 1997; 272(14): 9287 - 9293. [Abstract] [Full Text] [PDF]

				N. B. E. Oldenburg, R. B. Evans-Storms, and J. A. Cidlowski In Vivo Resistance to Glucocorticoid-Induced Apoptosis in Rat Thymocytes with Normal Steroid Receptor Function in Vitro Endocrinology, February 1, 1997; 138(2): 810 - 818. [Abstract] [Full Text] [PDF]

				M. Grande, I van der Kraan, L de Jong, and R van Driel Nuclear distribution of transcription factors in relation to sites of transcription and RNA polymerase II J. Cell Sci., January 8, 1997; 110(15): 1781 - 1791. [Abstract] [PDF]

				T. Trapp and F. Holsboer Nuclear Orphan Receptor as a Repressor of Glucocorticoid Receptor Transcriptional Activity J. Biol. Chem., April 26, 1996; 271(17): 9879 - 9882. [Abstract] [Full Text] [PDF]

				R. H. Oakley, M. Sar, and J. A. Cidlowski The Human Glucocorticoid Receptor beta Isoform J. Biol. Chem., April 19, 1996; 271(16): 9550 - 9559. [Abstract] [Full Text] [PDF]

				B van Steensel, M Brink, K van der Meulen, E. van Binnendijk, D. Wansink, L de Jong, E. de Kloet, and R van Driel Localization of the glucocorticoid receptor in discrete clusters in the cell nucleus J. Cell Sci., January 9, 1995; 108(9): 3003 - 3011. [Abstract] [PDF]