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J. Biol. Chem., Vol. 277, Issue 39, 36223-36232, September 27, 2002
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From the § Department of Pharmacology,
University of Michigan Medical School, Ann Arbor, Michigan
48109-0632 and the Glucocorticoid receptors (GRs) must heterocomplex
with hsp90 to have an open steroid binding cleft that can be accessed
by steroid. We reported that a seven-amino acid sequence (547-553 of
rat GR) overlapping the amino-terminal end of the ligand binding domain is required for hsp90 binding to GR. We have now
conducted saturation mutagenesis of this sequence, which appears to be
part of the surface where the ligand binding cleft merges with the surface of the ligand binding domain. No single point mutation causes
significant changes in any of a variety of biochemical and biological
properties in addition to hsp90 binding. A triple mutation
(P548A/T549A/V551A) increases by >100-fold the steroid concentration
required for half-maximal induction without affecting the level of
maximal induction or coactivator response. Interestingly, this triple
mutant displays reduced binding of steroid and hsp90 in whole cells,
but it possesses wild type affinity for steroid and normal hsp90
binding capacity under cell-free conditions. This phenotype of a
dramatic shift in the dose response for transactivation would be
expected from an increase in the rate of disassembly of the triple
mutant GR·hsp90 heterocomplex in the cell. Mutation of the entire
seven-amino acid region to CAAAAAC maintains the presence of a critical
Mutations at Positions 547-553 of Rat Glucocorticoid Receptors
Reveal That hsp90 Binding Requires the Presence, but Not Defined
Composition, of a Seven-amino Acid Sequence at the Amino Terminus of
the Ligand Binding Domain*
,,,¶
Steroid Hormones Section,
Laboratory of Molecular and Cellular Biology, NIDDK, National
Institutes of Health, Bethesda, Maryland 20892-0805
-helical structure and heterocomplex formation with hsp90 but
eliminates steroid binding and transcriptional activation, thus
disconnecting hsp90 binding from opening of the ligand binding cleft
and steroid binding.
*
This work was supported in part by National Institutes of
Health Grant DK31753 (to W. B. P.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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