Abstract
Previous studies have shown that the avian progesterone receptor, when in the nontransformed 8 S state, is complexed to another cellular protein having a molecular weight of 90,000. In this report, we show that this receptor-binding protein is indistinguishable from the 90,000-dalton protein which associates in a complex with the Rous sarcoma virus transforming protein, pp60v-src. This identity was established by the following criteria. 1) Monoclonal antibodies directed against the pp60v-src-associated 90-kDa protein recognized the 90-kDa progesterone receptor binding protein in an immunoblot assay. Conversely, monoclonal antibodies that recognize the progesterone receptor binding protein bind to the 90-kDa protein which complexes with pp60v-src. 2) Peptide maps prepared from the 90-kDa proteins immunoprecipitated from chicken cells with monoclonal antibodies directed against either the 90-kDa receptor binding protein or the 90-kDa pp60v-src-associated protein were indistinguishable. 3) Preincubation of the progesterone receptor complex with monoclonal antibodies prepared against the pp60v-src-associated protein caused a shift in the sedimentation of the progesterone receptor. Previous studies have established that the pp60v-src-associated protein is indistinguishable from one of the major heat shock proteins which are induced under a variety of stress conditions in eukaryotic cells. These present studies implicate a new role for this 90-kDa protein in the action of steroid hormones.
Highlights
From the $Department of Microbiology, State University of New York a t Stony Brook, Stony Brook, New York 11794 and the §Department of Cell Biology, Mayo Medical School, RochesteMr, innesota 55905
1) Monoclonal antibodies directed against the pp60"s'e-associated90-kDa protein recognized the 90-kDa progesterone receptor binding protein in an immunoblot assay
Plex with monoclonal antibodies prepared against the Theapparent similarities between the 90-kDa protein pp60""'"-associated protein caused ashift in the sedi- which associates with pp6OV~"a'"nd thesteroid receptor bindmentation of the progesterone receptPorre. viousstud- ing protein led us to investigate the possibility that they were ies have established that the pp60"""-associated pro- the same protein
Summary
Cepko et al [24]. This monoclonal antibody has been shown previously to recognize the pp6OV'"".pp90.pp complex [9, 12]. Immunostaining of proteinson nitrocellulosewasperformed as descrihed previously [4] This involved incuhation of cellulose strips with the various antihodies (approximately A0 pg for 5 h at 23 "C), washing, and incuhation with rahhit anti-mouse IgG antibody conjugated with alkaline phosphatase (Sigma)for 1 h or with "'1-protein. The cytosol was incuhated with 12 nM ["Hlprogesterone and p M cortisol for 1 h in ice. Unhound steroid wasremoved hv incuhating thecytosol for 1 0 min in ice with a pellet of charcoal derived from a n equal volume of 0.1% charcoal, 0.01% dextran in the same huffer.After centrifugation, cytosol aliquots (50 pl) were incuhated with 50 p1 of antihody plus 100 ~1 of huffer for 2 h in ice. The samples were layered on 5-20% sucrosegradientscontaining 10 mM potassium phosphate, 10 mM thioglycerol, and 10 mM sodium rnolyhdate, pH 7, and centrifugedfor 16 h a t 105,000X g. Fractionated, and measured for radioactivity as descrihed previously [4]
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