Using dosage suppression analysis in yeast cells to isolate factors involved in ER signal transduction, we have identified the yeast homologue of the human p23 (yhp23) as a protein that, when overexpressed, results in a 10-fold increase in G400V ER transcriptional activation. In vivo estradiol-binding assays suggest that yhp23 overexpression increases G400V ER transcriptional activity by increasing the number of estradiol-bound receptors. The effect of yhp23 overexpression was not limited to G400V ER, as it also increases both ligand binding and transcriptional activation by wt ER. No effect of yhp23 overexpression was observed on the constitutive activity of ER1–115, thereby demonstrating that yhp23 does not affect AF-1 activity per se. We therefore conclude that yhp23 is a member of the reconstituted steroid receptor signaling pathway in yeast cells, acting at the step of ligand binding by the receptor. This role is consistent with the currently proposed function of human p23 in steroid receptor-aporeceptor complex formation.
By using a yeast strain deficient in yhp23 expression (KO), analyses of wt ER signaling as a function of yhp23, wt ER, and estradiol concentrations were carried out. Our studies demonstrate that the magnitude of the effect of yhp23 on wt ER transcriptional activation is inversely proportional to the concentration of both wt ER and estradiol. Thus, at low, subsaturating concentrations of estradiol, yhp23 overexpression markedly increases wt ER transcriptional activation. In contrast, at saturating concentrations of estradiol, the effect of yhp23 overexpression on wt ER transcriptional activation is comparatively small. Furthermore, the magnitude of the effect of yhp23 on wt ER transcriptional activation is greater at low, rather than high, wt ER expression levels. Taken together, our findings indicate that the effect of yhp23 on wt ER signaling varies depending on yhp23, wt ER, and estradiol concentrations.
Subcellular localization studies with a yhp23-GFP fusion protein indicate that yhp23 is largely cytoplasmic in the absence of ER expression. When coexpressed with either the mutant or wt ER, yhp23 colocalizes with the receptor to the nucleus in the absence of estradiol. This colocalization is reversed upon estradiol treatment, such that yhp23 is released into the cytoplasm. From these observations, we conclude that yhp23 is part of the ER aporeceptor complex in yeast cells and that the distribution of yhp23 within the cell is dynamic and affected both by ER expression and estradiol binding.
It has been proposed that the function of p23 in steroid receptor signaling is to promote, through its interaction with Hsp90, the maturation or stabilization of the aporeceptor complex (24
). This model, derived largely from in vitro experiments (45
), proposes that the heat shock proteins Hsp90, p60, Hsp70, and possibly Hsp40 (10
) form a complex termed a “foldosome” (23
), within which Hsp90 exists in a conformation incompatible with p23 binding (53
) (Fig. ). The foldosome binds to the free receptor, which exists in a conformation with low affinity for the ligand. In a process that requires ATP and monovalent cations, the Hsp90 component of the foldosome and the receptor undergo conformational changes (13
), such that Hsp90 is now capable of binding p23 (53
), and the receptor exhibits high-affinity steroid binding. p23 binding to Hsp90 appears to stabilize this immature aporeceptor complex in vitro. In the absence of p23, the Hsp90-receptor complex is inherently unstable and rapidly dissociates (13
FIG. 9 A model for p23-dependent and -independent ER signal transduction. A simplified version of the current model of aporeceptor complex formation as deduced from in vitro studies is indicated by the black arrows. Hsp90 refers to the Hsp90 dimer, 70 refers (more ...)
The ability of yhp23 to increase ER transcriptional activation might be expected if yhp23 is limiting for the formation of mature aporeceptor complexes. Although we cannot exclude the possibility that yhp23 overexpression increases the ratio of yhp23 binding to each molecule of ER, this idea runs contrary to the current model of p23 function. Increasing the concentration of yhp23, therefore, would be expected to result in a greater number of mature ER aporeceptor complexes in the cell. As a result, the total estradiol binding will increase, which is consistent with our in vivo estradiol binding assays (Fig. ). Thus, overexpression of yhp23, by increasing the number of mature aporeceptor complexes, will manifest itself as an increase in transcriptional activation by ER at a given hormone concentration (Fig. and ). Thus, our in vivo findings are consistent with the current model of p23 function as derived from in vitro experiments.
Our results indicating that wt ER activity is detectable in the absence of yhp23 (Fig. ), however, suggest that in addition to the p23-dependent pathway, there must also exist a p23-independent pathway leading to estradiol binding and signaling by the ER. We propose that p23-independent activation of the ER in vivo occurs through estradiol binding directly to the immature (p23-deficient) aporeceptor complex (Fig. ). This hypothesis is consistent with in vitro observations that in the absence of p23, the foldosome proteins Hsp90, Hsp70, and p60 are sufficient to induce the hormone-binding conformation of steroid receptors (13
). We therefore propose that estradiol binding by the ER is a composite of both the p23-independent and p23-dependent pathways. The relative contribution of each pathway to ER activation is dependent upon the concentration of p23, ER, and estradiol (see below).
One prediction of our model is that yhp23 becomes less relevant to ER activation as the ratio of immature to mature aporeceptor complexes increases. The ratio of the two types of aporeceptor complexes is, in turn, a reflection of both ER and yhp23 concentrations. Increasing ER expression when p23 levels are constant results in a greater number of the immature aporeceptor complexes, which favors hormone binding through the p23-independent pathway. Conversely, increasing yhp23 levels facilitates the formation of mature aporeceptor complexes and therefore the p23-dependent pathway. This model of ER signal transduction is consistent with our in vivo findings that indicate that the magnitude of the effect of yhp23 on ER transcriptional activation is inversely proportional to the concentration of ER (Fig. D).
The proposed model further envisions that the concentration of estradiol also affects the relative contribution of the p23-dependent and p23-independent steroid binding pathways. As suggested by Fig. , free steroid can be considered to be competing with yhp23 for binding to the immature aporeceptor complex. As a result, the p23-independent pathway becomes more prominent as estradiol concentrations rise or, conversely, as yhp23 levels fall. Consistent with this notion, our data demonstrate that the magnitude of yhp23’s effect on ER signaling is greatest at low subsaturating, rather than high saturating, estradiol concentrations (Fig. E). Thus, our model predicts that the balance among yhp23, ER, and estradiol, ultimately determines the relative contributions of the p23-dependent and p23-independent pathways to ER signal transduction.
Our model also provides insight into the observation that yhp23 overexpression induces G400V ER activity to a greater extent than that observed for wt ER (Fig. ). We suggest that by altering the steroid-binding domain conformation, the G400V mutation largely eliminates the p23-independent pathway. G400V ER is temperature sensitive, relative to the wt ER, for estradiol binding in vitro, displaying reduced estradiol binding at 25°C but not at 4°C (54
). This suggests that the G400V ER mutation destabilizes the conformation of the steroid-binding domain, such that the receptor is unable to bind steroid with high affinity at 25°C. This mutation does not inhibit G400V ER’s interaction with the aporeceptor complex since G400V ER has been reported to be complexed with Hsp90 (1
) and since our subcellular localization studies suggest that G400V ER associates with yhp23 as efficiently as does wt ER. We therefore propose that the G400V mutation, by altering the structure of the steroid-binding domain, largely inhibits estradiol binding to the transient, immature aporeceptor complex, thereby diminishing estradiol binding through the p23-independent pathway. As a result, yhp23 competes more effectively with estradiol for binding to the complex, favoring the p23-dependent estradiol-binding pathway. The stability gained through yhp23 binding to the Hsp90-chaperone machinery, in turn, facilitates steroid binding by G400V ER.
It has been suggested by others that wt ER does not stably interact with Hsp90. This conclusion is based, in part, on studies with a VP16-Gal-ERLBD
fusion protein (1
). Although these constructs continue to exhibit ligand-dependent activation, association of this construct with Hsp90 could not be demonstrated. While there are a number of possible reasons for this discrepancy, we feel that the most probable explanation is a difference in the inherent characteristics of the wt and fusion proteins (1
). In addition, there are several reports that support a role for Hsp90 in the process of ligand binding by ER. (i) ER has been isolated with members of the aporeceptor complex from MCF-7 cells (52
) and bovine uterus (49
). (ii) Hsp90 has been demonstrated to colocalize with ER in the nucleus (38
). (iii) ER function has been found to be affected by mutant Hsp90 molecules (44
). (iv) ER function is also affected by the Hsp90-specific inhibitor geldanamycin (51
). Although these studies do not exclude the possibility of additional transcriptional repressors of wt ER, these findings, along with our results, strongly support a role for the components of the Hsp90-based chaperone complex in ER signal transduction.
Elegant genetic studies of yhp23 function in yeast cells by Bohen (2
) and Fang et al. (16
) demonstrate that yhp23 associates with Hsp90 and is a part of the GR aporeceptor complex in yeast cells. In contrast to our findings with ER, analysis of androgen receptor (AR) signaling in yeast cells suggests that it is largely p23 independent. This may reflect inherent differences in the mechanism of signal transduction employed by the receptors. Alternatively, we would suggest that, although the analysis of AR signaling was performed under a range of steroid concentrations, the levels of AR expression used may have favored the p23-independent pathway. It would be interesting to reevaluate AR signaling as a function of yhp23 at both low AR and testosterone concentrations, conditions that would favor the p23-dependent pathway.
The partial complementation of human p23 in yeast cells lacking yhp23 strongly suggests that yhp23 functions as the p23 homologue with respect to ER signaling (Fig. ). Although the yeast and human p23 proteins have regions of identity, significant sequence differences between the proteins also exist (2
). We speculate that this reflects species-specific differences in p23-Hsp90 association and might therefore explain the inability of human p23 to fully complement the loss of yhp23 function. It would be interesting to examine whether yeast cells expressing human Hsp90 (44
) and p23 increase ER activity to the same extent as their yeast counterparts.
Finally, our studies also provide insight into the possible mechanisms by which the ER communicates with other signaling pathways. Unexpectedly, a significant increase in estradiol-independent activation of ER was observed as a result of yhp23 overexpression (Fig. F). This estradiol-independent activation of wt ER was also observed upon p23 overexpression in MCF-7 cells (Fig. ). Previous studies have proposed that estradiol-independent transcriptional activation by wt ER results, in part, from ER phosphorylation through an epidermal growth factor-dependent pathway (4
). Thus, the most direct interpretation of our data suggests that maintenance of ER within the aporeceptor complex facilitates (but is not essential to) estradiol-independent activity, perhaps by maintaining ER in a conformation amenable to phosphorylation.
Besides ER, several other signaling molecules (including c-Src and c-Raf) are dependent upon chaperone complexes for their function (45
). Yeast cells deficient in the DnaJ homologue YDJ1, for instance, display both altered steroid receptor and Src kinase activity (31
). Thus, molecular chaperones link diverse signaling pathways. Additional insight into the mechanism of this cross talk comes from our subcellular localization studies that reveal a striking colocalization of ER and yhp23 within the nucleus. Estradiol treatment was shown to liberate yhp23 (and presumably other chaperone proteins) from the nucleus, allowing it to redistribute throughout the cytoplasm, where it can potentially interact with other signaling proteins. Although our colocalization studies were carried out under conditions of overexpression, we speculate that estradiol activation of ER may, through the release of chaperone components, modulate the activity of a variety of chaperone-dependent pathways. In light of p23’s role in stabilizing chaperone complexes, it is likely to play a key regulatory role in any such “chaperone signaling.”
In conclusion, we have provided evidence that yhp23 is a member of the ER signaling pathway and a positive regulator of ER function. We also suggest that at high ER and/or estradiol concentrations, conditions often present in the yeast system or during transient overexpression of ER in cultured mammalian cells, ER signaling occurs largely through a p23-independent pathway. Under low physiological concentrations of ER and estradiol, p23 is likely to be an important contributor to ER signaling. Our results also indicate that alterations in the level or subcellular distribution of p23 are potential mechanisms for modulating estradiol-dependent and -independent ER transcriptional activation. We are currently examining whether the p23 levels, the subcellular distribution, or the modification state fluctuates between normal and tumor cells, during cellular proliferation, during differentiation, or upon growth factor treatment. As aporeceptor complex formation is also believed to be important for ligand binding by GR, PR, and AR, p23 likely plays an important role in these pathways as well.