The p23 molecular chaperones act at a late step in intracellular receptor action to differentially affect ligand efficacies - PubMed (original) (raw)

Comparative Study

. 2000 Feb 15;14(4):422-34.

Affiliations

Comparative Study

The p23 molecular chaperones act at a late step in intracellular receptor action to differentially affect ligand efficacies

B C Freeman et al. Genes Dev. 2000.

Abstract

Multiple molecular chaperones, including Hsp90 and p23, interact with members of the intracellular receptor (IR) family. To investigate p23 function, we compared the effects of three p23 proteins on IR activities, yeast p23 (sba1p) and the two human p23 homologs, p23 and tsp23. We found that Sba1p was indistinguishable from human p23 in assays of seven IR activities in both animal cells and in yeast; in contrast, certain effects of tsp23 were specific to that homolog. Transcriptional activation by two IRs was increased by expression of any of the p23 species, whereas activation by five other IRs was decreased by Sba1p or p23, and unaffected by tsp23. p23 was expressed in all tissues examined except striated and cardiac muscle, whereas tsp23 accumulated in a complementary pattern; hence, p23 proteins might contribute to tissue-specific differences in IR activities. Unlike Hsp90, which acts on IR aporeceptors to stimulate ligand potency (i.e., hormone-binding affinity), p23 proteins acted on IR holoreceptors to alter ligand efficiencies (i.e., transcriptional activation activity). Moreover, the p23 effects developed slowly, requiring prolonged exposure to hormone. In vitro, p23 interacted preferentially with hormone-receptor-response element ternary complexes, and stimulated receptor-DNA dissociation. The dissociation was reversed by addition of a fragment of the GRIP1 coactivator, suggesting that the two reactions may be in competition in vivo. Our findings suggest that p23 functions at one or more late steps in IR-mediated signal transduction, perhaps including receptor recycling and/or reversal of the response.

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Figures

Figure 1

Figure 1

Mammalian cells contain p23 homologs with distinct tissue expression patterns. Immunoblot analysis of extracts prepared from indicated Mus musculus tissues. The p23 homologs or Hsp70 were detected with monoclonal antibodies as indicated.

Figure 2

Figure 2

p23 differentially affects the activity of GR, MR, TR, or AR in animal cells. The effects of p23 (●), tsp23 (♦), or Sba1p (█) on (A) GR, (B) MR, (C) TR, or (D) AR was measured in transiently transfected HeLa cells. For comparison, GR, MR, TR, or AR was transfected in the absence of an exogenous p23 homolog (○). (E) As a control, the activity of c-Jun alone or cotransfected with SBA1, p23, or tsp23 was examined.

Figure 3

Figure 3

p23 homologs interact with IRs and affect their activities. (A) Effects of p23, tsp23, or Sba1p on the transcriptional activities of IRs in S. cerevisiae strain (YNK233; Δp23) bearing a disrupted SBA1 gene. The Δp23 strain carried expression plasmids for GR, PR, MR, ER, or TR and were cotransformed with a LEU2 marker plasmid (open bar) or an expression vector for Sba1p (solid bar), p23 (stippled bar), or tsp23 (hatched bar). All transformants carried a reporter plasmid with the appropriate response elements driving a CYC1–lacz fusion. Data were normalized to the activity of the Δp23 strain carrying the LEU2 marker plasmid and represent the average values from three independent assays; error bars,

s.e.m.

. (B) Association of Sba1p (s), p23 (p), tsp23 (ts), and Hsp82 with IRs. Following growth in the appropriate selective medium and exposure to 10 μ

m

corticosterone where indicated, cell extracts were prepared by glass-bead homogenization and clarification by centrifugation. The GR, MR, or control protein c-Jun was precipitated from 250 μg of protein extract using antibodies specific for each factor. The reactions were subjected to 12% SDS-PAGE, electroblotted to Immobilon-P, and the indicated proteins detected by immunoblotting.

Figure 4

Figure 4

The molecular chaperone p23 differentially alters the activity of intracellular receptors. The dose response behavior of GR (A), PR (B), MR (C), ER (D), AR (E), TR (F), or RAR (G) in the parent (wild-type) and p23 disruption strain (Δp23) with (wild-type █; Δp23 ●) or without (wild-type □; Δp23 ○) plasmid expression of Sba1p. Data represent average values from three independent assays; error bars,

s.e.m.

. (H) Western blots from yeast extract preparations are shown; sample order is wild type, wild type with plasmid expressing Sba1p, Δp23, and Δp23 with plasmid expressing Sba1p. GR, AR, ER, TR, and Sba1p were detected by immunoblotting with polyclonal antibodies.

Figure 5

Figure 5

p23-mediated affects on ligand efficacy for GR and AR occur with slow kinetics in animal and yeast cells. The effect of p23 (circles) or tsp23 (squares), at the indicated time points, on GR (open symbols) or AR (closed symbols) was determined in transiently transfected (A) HeLa cells. The data are presented as the fold change in receptor activity relative to receptor activity in the absence of a cotransfected p23 or tsp23 expression plasmid. The activity of GR (open symbols) or AR (closed symbols) was measured in (B) wild-type yeast (circles), Δp23 expressing exogenous Sba1p (squares), and wild-type expressing exogenous Sba1p (diamonds) at the indicated time points. The data are presented as the fold change in receptor activity relative to receptor activity in the Δp23 strain.

Figure 6

Figure 6

Point mutations in hsp82 alter ligand potency for intracellular receptors. The activity of GR (A), MR (B), PR (C), and AR (D) was determined in the presence of wild-type Hsp82 (●) or the Hsp82 point mutants G313N (□), T525I (▵), or A576T/R579K (○).

Figure 6

Figure 6

Point mutations in hsp82 alter ligand potency for intracellular receptors. The activity of GR (A), MR (B), PR (C), and AR (D) was determined in the presence of wild-type Hsp82 (●) or the Hsp82 point mutants G313N (□), T525I (▵), or A576T/R579K (○).

Figure 6

Figure 6

Point mutations in hsp82 alter ligand potency for intracellular receptors. The activity of GR (A), MR (B), PR (C), and AR (D) was determined in the presence of wild-type Hsp82 (●) or the Hsp82 point mutants G313N (□), T525I (▵), or A576T/R579K (○).

Figure 6

Figure 6

Point mutations in hsp82 alter ligand potency for intracellular receptors. The activity of GR (A), MR (B), PR (C), and AR (D) was determined in the presence of wild-type Hsp82 (●) or the Hsp82 point mutants G313N (□), T525I (▵), or A576T/R579K (○).

Figure 7

Figure 7

In vivo hormone accumulation mediated by GR or MR is unaffected by expression levels of Sba1p. Wild-type (squares) or Δp23 (circles) carrying GR (A) or MR (B) expression plasmid, and either a Sba1p expression plasmid (closed markers) or LEU2 marker plasmid (open markers), were exposed to 0.1 μ

m

[3H]corticosterone for the indicated times at 22°C. Following extensive washing, the amount of accumulated [3H]corticosterone was quantified. As a control for nonspecific hormone accumulation, wild-type (▴) and Δp23 (▵) strains not expressing an IR were tested.

Figure 8

Figure 8

Sba1p differentially influences the activities GR and MR through the ligand-binding domain. The effect of Sba1p on GR–MR chimeric receptors, in wild-type (shaded bars), wild-type expressing Sba1p from a plasmid (solid bars), Δp23 (stippled bars), or Δp23 expressing Sba1p from a plasmid (hatched bars) was examined. Data for the chimeric receptors were normalized to the activity of the parent strain containing endogenous levels of Sba1p (three independent assays); error bars,

s.e.m.

.

Figure 9

Figure 9

p23 inhibits the DNA-binding activity of human TRα in vitro. (A) Purified recombinant (1.5 μg) Hsp90, TRα, p23, and tsp23 visualized by Coomassie blue. Anisotropy values for fluorescein-TREpal oligonucleotide bound by TR upon titration of p23 (B) or tsp23 (C). (D) The disassociation constant (Kd) between TR–p23 was determined by fitting a curve to the absolute value of the change in anisotropy as a function of p23 concentration (see Materials and Methods).

Figure 10

Figure 10

The NR-box 2 peptide from GRIP1 prevents and reverses p23-mediated inhibition of the TR–DNA interaction. (A) Wild-type NR-box 2 peptide (●; KHKLHRLLQDSS) or a mutant NR-box 2 peptide (○; KHKLHRAAQDSS) was added at the indicated concentrations into a reaction containing fluorescein-TREpal oligonucleotide, TR and p23 (10 μ

m

), and anisotropy was measured. (B) Aliquots of p23 and NR-box 2 peptide were added sequentially to a reaction mix of fluorescein-TREpal oligonucleotide and TR; anisotropy was measured after each addition.

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