Binding sites in the nuclear envelope for cytoplasmic glucocorticoid receptor complex

Using tritiated triamcinolone acetonide to monitor purification, cytoplasmic triamcinolone acetonide-receptor complex has been purified 3 000 fold from rat liver cytosol. The isolated complex sedimented as a single radioactive peak on a 5 - 20% sucrose gradient. Nuclear envelopes isolated from purified rat liver nuclei were found to contain binding sites for the partially purified cytoplasmic triamcinolone acetonide-receptor complex. The binding constants showed two saturable high affinity binding sites and the envelope bound the complex with a specific activity ten times higher than the plasma membrane and more than three times higher than the two endoplasmic types of membrane. Saturable binding to chromatin was not observed in the concentration range tested. Free steroid hormone did not bind the envelope. Binding sites for steroid hormones or steroid hormone-receptor complexes have been demonstrated both in chromatin and the nuclear protein matrix (Barrack and Coffey, 1980; Spelsberg, 1976). Because the nuclear envelope may be isolated with both these nuclear subfractions, the observed binding sites for steroid hormone-receptor complexes might be due to the presence of envelope components. The extent of association of nuclear envelope or nuclear envelope components with chromatin and the matrix was therefore investigated. Nuclear envelope fragments could be isolated from chromatin purified by centrifugation through 1,7 M sucrose. The binding of triamcinolone acetonide-receptor complex to these fragments was indistinguishable from the binding to purified nuclear envelope. A certain class of saturable chromatin binding sites for steroid hormone-receptor complexes may thus be due to the presence of envelope fragments. Extensive association of nuclear envelope polypeptides with the nuclear protein matrix was also observed. The matrix however, failed to bind triamcinolone acetonide-receptor complex. The nuclear envelope comprises an inner and outer membrane with well defined pore complexes spanning both membranes. In order to identify the location of the binding sites for trimacinolone acetonide-receptor complex in the envelope, fractionation and reconstitution of envelope proteins and lipids was attempted. Envelopes were solubilized in 2 - chloroethanol and protein and lipid components separated by chromatography on Sephadex LH 20. Envelope protein and lipid could be successfully reconstituted from chloroethanol by dialysis against aqueous buffer. Results showed that the receptor complex binds to the protein rather than lipid component of the envelope. This component was extractable by concentrations of the nonionic detergent Triton X-100 which do not extract the pore complex or lamina components of the envelope and is therefore probably a loosely bound membrane protein. The presence of specific binding sites for triamcinolone acetonidereceptor complex on the nuclear envelope may be necessary for the transport of the complex into the nucleus. The possibility that the envelope mediates the glucocorticoid response in ways not linked to transport of the cytoplasmic receptor complex into the nucleus cannot be ruled out.

Grantor dc:publisher.institution

Department of Molecular and Cell Biology

Year dc:date.issued

1983

Author dc:creator

• Smith, Peter John

Advisor dc:contributor.advisor

• Von Holt, Claus

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/11427/21908

OAI identifier oai:identifier

oai:open.uct.ac.za:11427/21908