Functional Comparison of Human and Drosophila Hop Reveals Novel Role in Steroid Receptor Maturation

Michael Chinkers,David F. Smith,Patricia E. Carrigan,Daniel L. Riggs

doi:10.1074/jbc.m414245200

Michael Chinkers, David F. Smith + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m414245200

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Abstract

Hsp70/Hsp90 organizing protein (Hop) coordinates Hsp70 and Hsp90 interactions during assembly of steroid receptor complexes. Hop is composed of three tetratricopeptide repeat (TPR) domains (TPR1, TPR2a, and TPR2b) and two DP repeat domains (DP1 and DP2); Hsp70 interacts directly with TPR1 and Hsp90 with TPR2a, but the function of other domains is less clear. Human Hop and the Saccharomyces cerevisiae ortholog Sti1p, which share a common domain arrangement, are functionally interchangeable in a yeast growth assay and in supporting the efficient maturation of glucocorticoid receptor (GR) function. To gain a better understanding of Hop structure/function relationships, we have extended comparisons to the Hop ortholog from Drosophila melanogaster (dHop), which lacks DP1. Although dHop binds Hsp70 and Hsp90 and can rescue the growth defect in yeast lacking Sti1p, dHop failed to support GR function in yeast, which suggests a novel role for Hop in GR maturation that goes beyond Hsp binding. Chimeric Hop constructs combining human and Drosophila domains demonstrate that the C-terminal domain DP2 is critical for this previously unrecognized role in steroid receptor function.

Highlights

Functional maturation of steroid receptors requires multistep assembly with molecular chaperones [1], and Hsp90 organizing protein (Hop), which binds both Hsp70 and Hsp90, can facilitate the progression through the intermediate stages of assembly [2]
Sti1p and human Hop (hHop) share over 50% amino acid sequence similarity and have a common domain structure consisting of three tetratricopeptide repeat domains (TPR1, TPR2a, and TPR2b) and two small domains (DP1 and DP2) containing a characteristic DP repeat motif [17]
Antibodies—The following mouse monoclonal antibodies were used in these studies: F5 [28], which recognizes vertebrate Hop; St2 [15], which is specific for yeast Sti1p; PR22 [29], which recognizes the progesterone receptor (PR); BuGR2 (Affinity Bioreagents, Golden, CO), which recognizes the glucocorticoid receptor (GR); and anti-L3, which reacts with a yeast ribosomal protein [30]

Summary

Introduction

Functional maturation of steroid receptors requires multistep assembly with molecular chaperones [1], and Hop, which binds both Hsp and Hsp, can facilitate the progression through the intermediate stages of assembly [2]. Hop-Hsp complexes are recruited to bind the receptor-associated Hsp. We have shown [16] that hHop can fully rescue the growth phenotype in a yeast strain (sti1⌬0) lacking the gene for Sti1p and that hHop can fully restore steroid receptor function in the sti1⌬0 background. It appears that Sti1p and hHop are functionally interchangeable. Hop TPR1 is required for Hsp binding [20, 21], and the Hsp70-specific peptide PTIEEVD co-crystallizes with TPR1

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