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Expression and characterization of human FKBP52, an immunophilin that associates with the 90-kDa heat shock protein and is a component of steroid receptor complexes.

ABSTRACT: Using an FK506 affinity column to identify mammalian immunosuppressant-binding proteins, we identified an immunophilin with an apparent M(r) approximately 55,000, which we have named FKBP52. We used chemically determined peptide sequence and a computerized algorithm to search GenPept, the translated GenBank data base, and identified two cDNAs likely to encode the murine FKBP52 homolog. We amplified a murine cDNA fragment, used it to select a human FKBP52 (hFKBP52) cDNA clone, and then used the clone to deduce the hFKBP52 sequence (calculated M(r) 51,810) and to express hFKBP52 in Escherichia coli. Recombinant hFKBP52 has peptidyl-prolyl cis-trans isomerase activity that is inhibited by FK506 and rapamycin and an FKBP12-like consensus sequence that probably defines the immunosuppressant-binding site. FKBP52 is apparently common to several vertebrate species and associates with the 90-kDa heat shock protein (hsp90) in untransformed mammalian steroid receptor complexes. The putative immunosuppressant-binding site is probably distinct from the hsp90-binding site, and we predict that FKBP52 has different structural domains to accommodate these functions. hFKBP52 contains 12 protein kinase phosphorylation-site motifs and a potential calmodulin-binding site, implying that posttranslational phosphorylation could generate multiple isoforms of the protein and that calmodulin and intracellular Ca2+ levels could affect FKBP52 function. FKBP52 transcripts are present in a variety of human tissues and could vary in abundance and/or stability.

SUBMITTER: Peattie DA

PROVIDER: S-EPMC50465 | biostudies-other | 1992 Nov

REPOSITORIES: biostudies-other

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Expression and characterization of human FKBP52, an immunophilin that associates with the 90-kDa heat shock protein and is a component of steroid receptor complexes.

Peattie D A DA Harding M W MW Fleming M A MA DeCenzo M T MT Lippke J A JA Livingston D J DJ Benasutti M M

Proceedings of the National Academy of Sciences of the United States of America 19921101 22

Using an FK506 affinity column to identify mammalian immunosuppressant-binding proteins, we identified an immunophilin with an apparent M(r) approximately 55,000, which we have named FKBP52. We used chemically determined peptide sequence and a computerized algorithm to search GenPept, the translated GenBank data base, and identified two cDNAs likely to encode the murine FKBP52 homolog. We amplified a murine cDNA fragment, used it to select a human FKBP52 (hFKBP52) cDNA clone, and then used the c ...[more]

PMID: 1279700

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Project description:Two isoforms of the 90-kDa heat shock protein, HSP90alpha and HSP90beta, are present in the cytosol of mammalian cells. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions (native PAGE) revealed that HSP90alpha predominantly exists as a homodimer and that HSP90beta is present mainly as a monomer [Minami, Kawasaki, Miyata, Suzuki and Yahara (1991) J. Biol. Chem. 266, 10099-10103]. However, only the dimeric form has been observed under other analytical conditions such as gradient centrifugation. In this study, therefore, we investigated native forms of HSP90 by use of immunochemical techniques with isoform-specific monoclonal antibodies recently developed in our laboratory. Glycerol gradient centrifugation at the physiological salt concentration as well as native PAGE analysis of rat liver cytosol revealed oligomeric forms of HSP90alpha sedimenting at 8-10S as predominant ones. On the other hand, the glycerol gradient centrifugation revealed multiple forms of HSP90beta oligomers sedimenting at 6-12S. All of the HSP90beta oligomers, however, migrated at 100-kDa monomer and 190-kDa dimer positions on native PAGE. A novel two-dimensional double native PAGE revealed that the entity was converted from the HSP90beta dimer to monomers during the electrophoresis. The same PAGE further revealed that the HSP90alpha oligomer also dissociated into dimers during the electrophoresis. Full-length form of bacterially-expressed human HSP90alpha migrated as dimers, but a considerable amount did not penetrate into the gel under native PAGE conditions, indicating the existence of oligomeric forms. Electrophoretic studies of deletion mutants of HSP90 demonstrated that the C-terminal 200 amino acids were capable of forming oligomers. Taken together, we conclude that both of the HSP90 isoforms predominantly exist as oligomeric forms in the cytosol even under unstressed conditions but that they artificially dissociate into smaller forms when subjected to native PAGE.

Project description:We analysed the inhibitory effects in vitro and in vivo of several metal ions on aldosterone binding to the rat kidney mineralocorticoid receptor with the purpose of assessing possible toxic effects of those ions on sodium retention, as well as to obtain information on receptor structural requirements for ligand binding. For the assays in vitro, the inhibitory effects of 20 metal ions were analysed on steroid-binding capacity for renal receptor cross-linked to 90-kDa heat-shock protein (hsp90) by pretreatment with dimethyl pimelimidate. Cross-linking prevented the artifactual dissociation of hsp90 (and, consequently, the loss of steroid binding) from the mineralocorticoid receptor due to the presence of high concentrations of salt in the incubation medium. Cross-linked heterocomplex showed no difference in ligand specificity and affinity with respect to native receptor, but increased stability upon thermal- or ionic-strength-induced destabilization was observed. Treatments in vitro with metal ions in the range 10(-8)-10(-1) M resulted in a differential inhibitory effect for each particular ion on aldosterone binding. Using the negative logarithm of metal concentration for 50% inhibition, the ions could be correlated with their Klopman hardness constants. The analysis of this relationship led us to postulate three types of reaction: with thiol, imidazole and carboxyl groups. The essential role played by these residues in steroid binding was confirmed by chemical modification of cysteines with dithionitrobenzoic acid, histidines with diethyl pyrocarbonate and acidic amino acids with Woodward's reagent (N-ethyl-5-phenylisoxazolium-3'-sulphonate). Importantly, the toxic effects of some metal ions were also observed by treatments in vivo of adrenalectomized rats on both steroid-binding capacity and aldosterone-dependent sodium-retaining properties. We suggest that those amino acid residues are involved in the activation process of the mineralocorticoid receptor upon steroid binding. Thus toxic effects observed with these metal ions may be a consequence of modifications of those essential groups. Our results support the notion that toxicity of metals on renal mineralocorticoid function may be predicted according to their chemical hardness.

Dataset Information

Expression and characterization of human FKBP52, an immunophilin that associates with the 90-kDa heat shock protein and is a component of steroid receptor complexes.

Publications

Expression and characterization of human FKBP52, an immunophilin that associates with the 90-kDa heat shock protein and is a component of steroid receptor complexes.

Similar Datasets

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