Rabbit FKBP59-heat shock protein binding immunophillin (HBI) is a calmodulin binding protein.

FKBP59-HBI, a heat shock protein hsp90-binding immunophilin that was originally detected in heterooligomer forms of steroid receptors, is retained on Calmodulin (CAM)-Sepharose 4B in the presence of 2 mM Ca2+ and is eluted by EGTA, demonstrating a specific p59-CAM interaction. The p59 amino acid sequence reveals the presence of two putative CAM binding sites in a helix regions of the protein, as well as PEST sequences which are generally present in CAM-binding proteins. In vitro proteolysis by calpain II (a Ca(2+)-activated neutral protease), another feature of CAM-binding proteins, generates shorter peptides revealed by the mAb EC1, but not by the pAb 173 which recognizes the C-terminal of the protein. The potential function of CAM binding by the hsp90-binding immunophilin is discussed.

An immunophilin that binds M(r) 90,000 heat shock protein: main structural features of a mammalian p59 protein.

In the rabbit, a p59 protein included in the untransformed, non-DNA binding, "8-9S," steroid receptor complexes binds heat shock protein M(r) approximately 90,000 (hsp90). Sequence data [Lebeau, M. C., Massol, N., Herrick, J., Faber, L. E., Renoir, J. M., Radanyi, C. & Baulieu, E. E. (1992) J. Biol. Chem. 267, 4281-4284] and hydrophobic cluster analysis delineate, from the N terminus, two successive domains closely related to the immunosuppressant FK506 binding immunophilin FKBP (FK506 binding protein), consistent with recent purification of the human p56 immunophilin cognate protein by FK506 affinity chromatography [Yem, A. W., Tomasselli, A. G., Heinrikson, R. L., Zurcher-Neely, H., Ruff, V. A., Johnson, R. A. & Deibel, M. R., Jr. (1992) J. Biol. Chem. 267, 2868-2871]. The first FKBP-like domain demonstrates all structural characteristics known to be necessary for immunosuppressant binding and for peptidylprolyl cis-trans isomerase (rotamase) activity. Hence, p59 is a "hsp binding immunophilin" (HBI). It is thus speculated that hsp binding immunophilin may help the assembly/disassembly mechanisms involved in steroid receptor trafficking and activity and participate in the poorly understood hsp90 function. ATP/GTP binding likely occurs within the second FKBP-like domain, near the FK506 binding site on the FKBP template. A third domain detected by the hydrophobic cluster analysis method is distantly structurally related to the two first FKBP-like domains and is followed by the C-terminal part of the protein, which contains a calmodulin binding consensus sequence. Hsp binding immunophilin may be involved in a number of immunological, endocrinological, and chaperone-mediated pathways.

P59, an hsp 90-binding protein. Cloning and sequencing of its cDNA and preparation of a peptide-directed polyclonal antibody.

The primary sequence of the rabbit liver cDNA coding for protein p59 has been determined. The protein binds to the 90-kDa heat shock protein (hsp 90) and is associated with it, including when hsp 90 participates in hetero-oligomeric complexes of untransformed mammalian steroid receptors that sediment at 8-10 S. The cloned cDNA codes for an open reading frame of 458 amino acids defining a yet unknown protein. However, 55% amino acid homology to peptidyl-prolyl isomerase is found between amino acids 41 and 137, suggesting rotamase activity for p59, which speculatively may apply to bound hsp 90 and thus be implied in the intracellular trafficking of hetero-oligomeric forms of steroid hormone receptors. A polyclonal antibody derived from the COOH-terminal peptide 441-458 demonstrates a good affinity for rabbit, rat, and human "p59" protein. It interacts with at least one epitope, available in 8-10 S untransformed steroid receptor complexes and different from that recognized by the monoclonal antibody KN382/EC-1.

Interaction of oestrogen and progesterone receptors with specific subfractions of laying-hen oviduct chromatin.

Salt (NaCl)-extracted nuclear oestrogen receptor from hen oviduct was incubated with salt-depleted oviduct chromatin and dialysed to low salt. The oestrogen receptor (re)associated with chromatin to form a 13-14S-sedimenting fraction, as found in 'native' chromatin, and saturation of this interaction was obtained for very low receptor concentrations (approx. 0.04 nM). Similarly, purified progesterone receptor from chick oviduct cytosol associated with depleted chromatin to form an 11-12S-sedimenting fraction, as in 'native' chromatin; this interaction tended towards saturation for much higher concentrations of progesterone receptor (approx. 8 nM) than that observed for oestrogen receptor. When the two receptors were incubated with depleted chromatin from hen kidney or erythrocytes, their s values were as for oviduct chromatin. However, no saturation of these interactions was seen, even for high concentrations of receptor. Steroid-hormone receptors can therefore bind in vitro to particular subfractions of non-target-tissue chromatin, but with a much lower affinity than to target-tissue chromatin.

Oestrogen and progesterone receptors in chick oviduct chromatin after administration of oestradiol, progesterone or anti-oestrogen.

Oestrogen-primed and withdrawn chicks were injected with oestradiol benzoate, progesterone, and/or the anti-oestrogens tamoxifen and 4-hydroxytamoxifen. Oestrogen receptors were studied in oviduct chromatin solubilized by mild digestion of purified nuclei with micrococcal nuclease. After a single injection of oestradiol benzoate, ultracentrifugation on sucrose gradients of chromatin extracts labelled with [3H]-oestradiol showed two peaks of oestradiol binding sites, sedimenting at 13--14 S and 7--8 S. After repeated injections of oestradiol benzoate, the 13--14 S peak increased more than the 7--8 S peak. After injection of anti-oestrogen alone or together with oestradiol benzoate, no [3H]oestradiol-binding or 4-hydroxy[3H]tamoxifen-binding peaks were detected in the chromatin. Injection of progesterone also produced an increase of the 13--14 S and 7--8 S chromatin oestradiol receptor. Progesterone receptor could only by detected in chromatin early after progesterone administration, and it sedimented in density gradients with the 12 S mononucleosome fraction. Tamoxifen injected together with progesterone gave higher levels of 13--14 S oestrogen binding sites than did progesterone alone. The presence of a 13--14 S peak of oestrogen binding sites in hormonal situations which promote a biological response in the chick oviduct, and the absence of this peak after administration of anti-oestrogens, suggest that this subfraction of chromatin contains elements involved in gene regulation.

[Production and detection of antibody against estradiol receptor in the calf uterus. Interaction with the estrogen receptor from the hen oviduct]. / Production et détection d'anticorps antirécepteur de l'oesstradiol d'utérus de veau. Interaction avec le récepteur d'oviducte de poule.

The antibodies against estrogen receptor were obtained after injecting Rabbits with a cytoplasmic receptor fraction isolated from Calf uterus. The estrogen receptor was partially proteolysed by the action of trypsin and subsequently purified by affinity chromatography (purification 4,000 to 10,000 fold, to a purity of 5-20%). The affinity of the antibody for the proteolysed receptor is KD approximately 1 nM and serum titres have reached values of approximately 50 nM. The values remained constant after the third injection. Preliminary results indicate that the antibody has approximately the same affinity for "native" cytoplasmic estrogen receptor from Calf uterus, as well as for the "trypsinized" forms of estrogen receptor isolated from Calf uterine cytosol and Hen oviduct nuclei.

Estrogen receptor in hen oviduct chromatin, digested by micrococcal nuclease.

Nuclei from laying hen oviduct were prepared according to Hewish and Burgoyne i.e. in the presence of spermine and spermidine and in the absence of divalent cations and were then moderately digested by micrococcal nuclease. When the resulting chromatin was analysed by ultracentrifugation on a sucrose gradient, a peak of specific estradiol-binding sites was observed, sedimenting slightly faster (13-14 S) than the mononucleosomes (12 S). When the chromatin was centrifuged on a gradient containing heparin (5 microngram/ml) the sedimentation coefficient of the estradiol receptor peak shifted to 7-8 S; it returned to the 13-14 S position in the absence of heparin, when target organ chromatin was also present in the gradient. The preparation of the chromatin is described and the validity of the method to explore receptor localisation is discussed, as is the specificity of the receptor-DNA interaction.