The nuclear prolactin receptor: a 62-kDa chromatin-associated protein in rat Nb2 lymphoma cells.
Previously we demonstrated that lactogen-dependent Nb2 cells express a nuclear prolactin (PRL) receptor. Thus, the nuclear receptor expressed in PRL-dependent Nb2-11 and -independent Nb2-SFJCD1 cells was characterized. Initially, the potential proteolytic processing of internalized 125I-rPRL was investigated. Radiolabeled hormone eluted from a Sephadex G-100 column with a retention time identical to that found for stock hormone, indicating that nuclear PRL was intact. Experiments to investigate the nuclear distribution of the hormone demonstrated that 90% of 125I-rPRL bound to chromatin; the remaining was distributed between "sap-protein" and nucleoplasmic fractions. Chromatin-bound PRL was resistant to high salt and detergent extraction indicating a tight association. Immunoprecipitation and immunoblot analysis revealed the PRL receptor to be 62 kDa in each cell line. Affinity crosslinking experiments and immunoprecipitation demonstrated that 125I-rPRL complexed with a protein(s) of similar M(r) in intact cells. 125I-rPRL binding was saturable and of high affinity (Kds of 180 and 170 pM, for Nb2-11 and Nb2-SFJCD1 lines, respectively). PRL binding was competitively inhibited by ovine and bovine PRLs and hGH, but not by rat GH, and by monoclonal antibodies (McAbs) which recognize the lactogen binding site. These results demonstrate that: (1) 125I-rPRL translocates intact to the Nb2 cell nucleus and tightly associates with chromatin; (2) the chromatin receptor specifically binds 125I-rPRL with high affinity; and (3) the chromatin receptor is essentially identical to its membrane counterpart with respect to mass, binding characteristics, and McAb recognition.
Rao
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YP
Department of Pharmacology and Toxicology, University of North Dakota School of Medicine, Grand Forks 58202, USA.
Buckley
D J
DJ
Buckley
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AR
D000818:Animals; D001667:Binding, Competitive; D001692:Biological Transport; D002451:Cell Compartmentation; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000378:metabolism*; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D015151:Immunoblotting; D008223:Lymphoma / Q000378:metabolism; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011233:Precipitin Tests; D011388:Prolactin / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000302:isolation & purification / Q000378:metabolism*; D018000:Receptors, Peptide / Q000378:metabolism; D011981:Receptors, Prolactin / Q000302:isolation & purification / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D002843:Chromatin; D003432:Cross-Linking Reagents; D009687:Nuclear Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D018000:Receptors, Peptide; D011981:Receptors, Prolactin; C028202:placental lactogen receptor; D011388:Prolactin