The steroid binding domain influences intracellular solubility of the baculovirus overexpressed glucocorticoid and mineralocorticoid receptors.
The expression and formation of the oligomeric steroid binding form of the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR) were further examined in the baculovirus expression system. Analysis of the steroid binding ability and accumulation of the GR and MR in recombinant baculovirus-infected insect cells revealed that only 0.35% of the total expressed receptors are actually assembled into cytoplasmic oligomeric receptor complexes. The majority of the overexpressed GR and MR, which amounts to 50 pg/cell, appear to self-aggregate and form insoluble aggregates which fractionate with the nucleus. We believe that this large amount of receptors far exceeds the limited amounts of hsp90, hsp70, and other cellular factors which are required to assemble the heteromeric receptor complex. Attempts to assemble the GR and the MR in vivo by coexpression of the receptors with hsp90 or hsp70 failed to cause any increase in the formation of the steroid binding receptor complex and also in preventing the aggregation of the receptors. On the other hand, in vitro incubation of monomeric GR or MR partially purified from the insoluble receptor aggregates with reticulocyte lysate resulted in complete reconstitution of the oligomeric receptor complex with a concomitant restoration of full steroid binding ability. These data suggest that interaction of hsp90 with the GR or the MR is complex and highly regulated and requires the participation of other cellular factors which are limited in insect cells but can be supplied in vitro by reticulocyte lysate.(ABSTRACT TRUNCATED AT 250 WORDS)
Alnemri
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ES
Department of Pharmacology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
Litwack
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D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001665:Binding Sites; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000235:genetics; D007700:Kinetics; D046911:Macromolecular Substances; D009036:Moths; D011965:Receptors, Glucocorticoid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018161:Receptors, Mineralocorticoid; D011987:Receptors, Steroid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013256:Steroids / Q000378:metabolism*; D014162:Transfection
D006360:Heat-Shock Proteins; D046911:Macromolecular Substances; D011965:Receptors, Glucocorticoid; D018161:Receptors, Mineralocorticoid; D011987:Receptors, Steroid; D013256:Steroids