Hsp70-RAP46 interaction in downregulation of DNA binding by glucocorticoidreceptor

Receptor-associating protein 46 (RAP46) is a cochaperone that regulates the transactivation function of several steroid receptors. It is transported i nto the nucleus by a liganded glucocorticoid receptor where it downregulate s DNA binding and transactivation by this receptor. The N- and C-termini of RAP46 are both implicated in its negative regulatory function. In metaboli c labelling experiments, we have shown that the N-terminus of RAP46 is modi fied by phosphorylation, but this does not contribute to the downregulation of glucocorticoid receptor activity. However, deletion of a sequence that binds 70 kDa heat shock protein (Hsp70) and the constitutive isoform of Hsp 70 (Hsc70) at the C-terminus of RAP46 abrogated its negative regulatory act ion. Surface plasmon resonance studies showed that RAP36 binds the glucocor ticoid receptor only when it has interacted with Hsp70/ Hsc70, and confocal immunofluorescence analyses revealed a nuclear transport of Hsp70/Hsc70 by the liganded receptor. Together these findings demonstrate an important co ntribution of Hsp70/Hsc70 in the binding of RAP46 to the glucocorticoid rec eptor and suggest a role for this molecular chaperone in the RAP46-mediated do downregulation of glucocorticoid receptor activity.