OPTIMAL LIGAND-BINDING BY THE RECOMBINANT HUMAN GLUCOCORTICOID RECEPTOR AND ASSEMBLY OF THE RECEPTOR COMPLEX WITH HEAT-SHOCK-PROTEIN-90 CORRELATE WITH HIGH INTRACELLULAR ATP LEVELS IN SPODOPTERA-FRUGIPERDA CELLS

The full-length human glucocorticoid receptor (hGR), overexpressed in Spodoptera frugiperda (Sf9) cells, associates with heat shock protein 90 (hsp90) and hsp70 and binds dexamethasone with high affinity. Bacul ovirus infection of Sf9 cells grown in TNM-FH medium results in the ra pid depletion of glucose from the medium within 24h. Noting a discrepa ncy between hGR protein levels and Ligand binding capacity in such cul tures, we hypothesized that the depletion of glucose from the medium c ould result in intracellular ATP depletion and consequently affect the ligand binding capacity of the recombinant hGR. Supplementation of th e Sf9 culture medium with additional glucose resulted in a three-fold increase in intracellular ATP levels, and a three-fold increase in H-3 -dexamethasone binding capacity, without altering the protein levels o f hGR, hsp90 or hsp70. However, more hsp90 co-immunoprecipitated with hGR from cells grown in glucose supplemented medium. Our data support the hypothesis that high-affinity ligand binding by hGR requires the A TP-dependent formation of the hGR:hsp90 heterocomplex. Besides having practical consequences for the production of recombinant GR and other related proteins, our findings could ultimately have relevance in dise ases such as diabetes mellitus. (C) 1997 Elsevier Science Ltd.