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. 2011 Jul;16(4):469-74.
doi: 10.1007/s12192-010-0254-2. Epub 2011 Jan 16.

Single-point Mutation in a Conserved TPR Domain of Hip Disrupts Enhancement of Glucocorticoid Receptor Signaling

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Single-point Mutation in a Conserved TPR Domain of Hip Disrupts Enhancement of Glucocorticoid Receptor Signaling

Sean P Place. Cell Stress Chaperones. .
Free PMC article

Abstract

The Hsp70-interacting protein Hip has been identified as a transient participant in the assembly of both glucocorticoid (GR) and progesterone receptor complexes. Although it has been difficult to identify a physiological role for Hip, it is believed to have intrinsic chaperoning properties and has been identified as a potential anti-apoptotic target of Granzyme B. In vitro assays have provided evidence that Hip may interact with GR complexes in an Hsp70 independent manner and can enhance the function of GR in hormone based reporter assays. In this study, a cDNA for human Hip was used in mutational analysis to map Hip function to critical structural elements. A single amino acid substitution (L211S) resulted in a loss of Hip function. This mutation also appears to disrupt the interaction of Hip with Hsp70 in vitro. Failure to recover Hip-L211S constructs in co-immunoprecipitation assays with an Hsp70 monoclonal antibody suggests that the mutation is unlikely to result in a misfolded substrate.

Figures

Fig. 1
Fig. 1
a Functional domains of Hip targeted for mutagenesis are indicated above the diagram with known regions of homology with other protein motifs indicated below the diagram. b Mapping of point mutations created during error prone PCR to the functional domains of wt Hip that were screened for loss of function
Fig. 2
Fig. 2
a Growth curves at 30°C were determined in the presence of 50 μg/ml l-canavine sulfate salt (Sigma) for wild-type (wt) Hip (asterisk), empty vector (filled diamonds), GR alone (empty diamonds), or a library of random Hip mutants generated by low-fidelity PCR of wt Hip cDNA and GR (all others) and co-expressed in YNK512 plasmids generated via homologous recombination. b Colonies displaying a fast growth phenotype in the presence of canavanine were assayed for enhancement of GR function using the hormone-dependent reporter assay as previously reported (Nelson et al. 2004). c For mutants that showed defects in the enhancement of GR signaling, mutant Hip cDNAs were isolated from colony PCR reactions and ligated to the yeast expression vector p423GPD and transformed into a separate GR reporter expression strain (DSY-1000) to verify this effect was due to defects in Hip function. Five independent transformants were compared with wt Hip for ability to enhance GR function. To determine the rate of reporter expression, β-galactosidase induction curves were first generated by plotting relative light units against the OD600 of the culture sample. Regression analysis of this linear portion of each data set yielded a best-fit line (typically, R2 > 0.98) the slope of which is the growth rate-normalized rate of β-galactosidase expression. For convenience, the reporter expression units are defined as the slope/100. d Western immunoblots—whole-cell extracts were fractionated by SDS-PAGE and transferred to polyvinylidene fluoride membrane for immunoblot analysis using the mouse monoclonal antibody 2 G6 (anti-Hip; 1:5,000 dilution) to verify the expression of full-length Hip constructs in yeast isolates displaying a loss of function
Fig. 3
Fig. 3
a Hormone reporter assay—β-galactosidase expression was used to assess the impact of individual point mutations (lined bars) on the ability of wt Hip (black bar) or empty vector (open bar) to enhance GR function. b Western immunoblots—whole-cell extracts were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membrane for immunoblot analysis using the mouse monoclonal antibody 2 G6 (anti-Hip; 1:5,000 dilution) to verify the expression of full-length Hip constructs in yeast transformed with Hip point mutants. c In vitro co-immunoprecipitation—the abilities of Hip point mutants to bind Hsp70 were assessed utilizing Hsp70 immobilized on an immunoaffinity resin. Anti-Hsp70 monoclonal antibody BB70 was adsorbed to protein G-Sepharose (Pharmacia Biotech, Piscataway, NJ) and used to immunoprecipitate Hsp70 from rabbit RL (1:1; from Green Hectares, Oregon, WI) that was supplemented with a radiolabeled Hip form. Each sample contained the same molar equivalent of radiolabeled Hip or Hip mutant in 100 μl RL with 10 μg BB70 on a 15-μl resin pellet. Proteins adsorbed to resin were separated by SDS-PAGE and visualized by Coomassie blue staining and autoradiography of the dried gel. Control antibody used to compare background binding for the Hsp70 forms was the anti-PR monoclonal antibody, PR22

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