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

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.Functional maturation of steroid receptors requires multistep assembly with molecular chaperones (1), and Hop, which binds both Hsp70 and Hsp90, can facilitate the progression through the intermediate stages of assembly (2). The early steps in assembling receptor/chaperone complexes are the binding of Hsp40 to a receptor monomer (3) followed by recruitment of Hsp70. Hop-Hsp90 complexes are then recruited to bind the receptor-associated Hsp70. In a transition that remains poorly understood, Hsp90 becomes directly associated with the receptor, and Hsp70 and Hop leave the complex. The Hsp90 co-chaperone p23 binds to Hsp90, stabilizing its association with the receptor, and immunophilin-related co-chaperones bind Hsp90 at a site vacated by Hop. Only when the receptor has achieved assembly with Hsp90 and p23 does it attain full hormone binding ability (4,5).Through its ability to simultaneously bind both Hsp70 and Hsp90, Hop serves as an adaptor to coordinate the recruitment of Hsp90 to intermediate receptor complexes containing Hsp70 (6). Hop can also inhibit Hsp90 ATPase, but it acts somewhat differently toward yeast or vertebrate Hsp90. Yeast Hsp90 has a much higher basal ATPase activity, and Sti1p, the Hop ortholog in Saccharomyces cerevisiae (7), inhibits this activity (8,9). In contrast, human Hsp90 has very low basal ATPase activity that can be stimulated by binding to substrate, and Hop is able to inhibit the client-stimulated ATPase (10). In some settings Hop can also affect Hsp70 ATPase. For example, Sti1p dramatically stimulates activity of the yeast Ssa family of Hsp70 proteins (11), although Hop was not observed to stimulate the ATPase activity of vertebrate Hsp70 (12). Unlike most other receptor-associated chaperone components, Hop does not possess independent chaperone activity by as determined by in vitro refolding assays (13,14), so it is ...

Independent Regulation of Hsp70 and Hsp90 Chaperones by Hsp70/Hsp90-organizing Protein Sti1 (Hop1)

“…shown that the loss of GR signaling in a yeast strain lacking Sti1p is restored by human Hop1 and that a mutation in Hop1 (K73E) that reduces Hop1 interaction with human Hsp70 reduces Hop1 function in this GR signaling (13,29). Thus, Hop1 and Sti1p are functionally conserved with regard to GR activation.…”

“…We also constructed STI1 alleles lacking domains known to be important for specific interactions of Sti1p with Hsp70 and Hsp90 (6,13,29). Lastly, we assessed the abilities of human Hop1, a specific Hop1 mutant (K73E) known to be weakened in its interaction with human Hsp70 (13) These phenotypes are similar to that of cells lacking Sti1p (Fig.…”

Increased expression of co-chaperone HOP with HSP90 and HSC70 and complex formation in human colonic carcinoma

“…Folding of the client proteins is considered to be assisted by HSP70 and HSP90 in a sequential manner under the control by HOP (Nollen and Morimoto 2002;Pratt and Toft 2003;Wegele et al 2004;Wegele et al 2006). The organized action of the two chaperones prevents misfolding and aggregation of proteins, and thus facilitates productive folding of proteins and formation of functional protein complexes (Carrigan et al 2005). Kamal et al (2003) indicated that HSP90 forms complexes with co-chaperones HOP and p23 in tumor tissues and cultured tumor (BTB474, MCF7, and Hs578t) cells (Kamal et al 2003).…”

“…For example, signaling protein kinases (e.g., Cdk4, Cdk6, and Raf family kinases) are stabilized by HSP90 in the presence of Cdc37 prior to the formation of functional complexes or final enzymatic forms (Grbovic et al 2006;Vaughan et al 2006). HSP90 associates with steroid hormone receptors (e.g., glucocorticoid receptor, androgen receptor, and estrogen receptor) and regulates the transport and maturation of the receptors (Carrigan et al 2005;Fiskus et al 2007). We have shown that the HSP70 and HSP90 chaperones strongly interact with a number of medical drugs (Ishida et al 2008;Miyazaki et al 2004;Otaka et al 2007).…”

Single-point mutation in a conserved TPR domain of Hip disrupts enhancement of glucocorticoid receptor signaling

“…In addition co-expression of rat Hsp70 with Hip inhibited rather than enhanced Hip function (Nelson et al 2004). In addition, Hop containing a point mutation that disrupts Hsp70 binding will also partially rescue GR function in the Sti1p deficient strain (Carrigan et al 2005). Therefore, Hip and Hop may both have Hsp70-independent roles in GR maturation.…”