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 expresse d in PRL-dependent Nb2-11 and -independent Nb2-SFJCD1 cells was charac terized. Initially, the potential proteolytic processing of internaliz ed I-125-rPRL was investigated. Radiolabeled hormone eluted from a Sep hadex G-100 column with a retention time identical to that found for s tock hormone, indicating that nuclear PRL was intact, Experiments to i nvestigate the nuclear distribution of the hormone demonstrated that 9 0% of I-125-rPRL bound to chromatin; the remaining was distributed bet ween ''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 ex periments and immunoprecipitation demonstrated that I-125-rPRL complex ed with a protein(s) of similar M(r) in intact cells. I-125-rPRL bindi ng was saturable and of high affinity (K(d)s of 180 and 170 pM, for Nb 2-11 and Nb2-SFJCD1 lines, respectively), PRL binding was competitivel y inhibited by ovine and bovine PRLs and hGH, but not by rat GH, and b y monoclonal antibodies (McAbs) which recognize the lactogen binding s ite, These results demonstrate that: (1) I-125-rPRL translocates intac t to the Nb2 cell nucleus and tightly associates with chromatin; (2) t he chromatin receptor specifically binds I-125-rPRL with high affinity ; and (3) the chromatin receptor is essentially identical to its membr ane counterpart with respect to mass, binding characteristics, and McA b recognition. (C) 1995 Academic Press, Inc.