Expansion of tropism of a feline parvovirus to target a human tumor cell line by display of an alpha(v) integrin binding peptide on the capsid.

The autonomous parvoviruses are small, non-enveloped, single strand DNA viruses. They occur in many species and they have oncolytic properties. We are modifying the capsid of feline panleukopenia virus (FPV), a parvovirus which normally infects feline cells, with the goal of targeting human tumor cells for potential cancer therapy. Using recombinant viruses transducing a luciferase reporter, we show that insertion of a cyclically constrained, integrin-binding peptide at an exposed position on the FPV capsid enables transduction of an alpha(v) integrin-expressing human rhabdomyosarcoma cell line (Rh18A). These cells were not transduced by virus with the unmodified FPV capsid. Transduction of Rh18A was specifically inhibited by an alpha(v) integrin blocking antibody. However, other human tumor lines expressing alpha(v) integrins were not transduced by virus with either the modified or unmodified capsid. We conclude that modification of the FPV capsid to bind alpha(v) integrins can contribute to, but is not generally sufficient for, redirecting infection to human tumor cells. The permissiveness of Rh18A cells presumably involves additional factors unique to this line among various human cell lines tested.

Regulation of glucocorticoid receptor function through assembly of a receptor-heat shock protein complex.

Incubation of immunopurified, hormone-free mouse glucocorticoid receptors with rabbit reticulocyte lysate results in ATP-dependent and monovalent cation-dependent assembly of the GR into a heterocomplex with hsp90, hsp70, and hsp56. Heterocomplex assembly is accompanied by conversion of the receptor from a form that does not bind steroid to a high affinity steroid-binding conformation. Reticulocyte lysate also promotes ATP-dependent dissociation of unliganded receptors from a prebound receptor-DNA complex. Receptor released from DNA has been reconstituted into the heat shock protein heterocomplex and converted to the non-DNA-binding state. The reticulocyte lysate also reconstitutes pp60v-src into a heterocomplex containing hsp90 and p50, both of which are components of the native heterocomplex form of the tyrosine kinase in cytoplasm. Although the c-Raf-1 serine/threonine kinase has never been found in native association with hsp90, it can be assembled into a heat shock protein heterocomplex by the ATP-dependent system in reticulocyte lysate.

Evidence that the hormone binding domain of steroid receptors confers hormonal control on chimeric proteins by determining their hormone-regulated binding to heat-shock protein 90.

Previously, it has been shown that the hormone binding domain of the glucocorticoid receptor acts as a transferable regulatory cassette that can confer hormonal control onto chimeric proteins [Picard, D., Salser, S. J., & Yamamoto, K. R. (1988) Cell 54, 1073-1080]. The hormone binding domain of the glucocorticoid receptor contains its site of interaction with the 90-kDa heat-shock protein, hsp90 [Dalman, F. C., Scherrer, L. C., Taylor, L. P., Akil, H., & Pratt, W. B. (1991) J. Biol. Chem. 266, 3482-3490]. We have now transfected COS cells with cDNAs for fusion proteins containing beta-galactosidase and portions of the glucocorticoid receptor, and we demonstrate a correlation between hormone regulation of fusion protein localization and binding of the fusion proteins to hsp90. The hormone binding domain (residues 540-795) of the rat glucocorticoid receptor is sufficient for conferring hormone regulation onto a fusion protein and for intracellular binding of a fusion protein to hsp90. The hormone binding domain of the rat glucocorticoid or the human estrogen receptor is also sufficient to permit reticulocyte lysate-mediated refolding of a fusion protein into association with hsp90. Consistent with the results of fusion protein localization in intact cells, binding of a fusion protein to hsp90 blocks binding of antibody directed against the NL1 nuclear localization signal of the glucocorticoid receptor. These observations argue strongly that the hormone binding domain of the glucocorticoid receptor confers hormonal control of fusion proteins by conferring hormone-regulated binding to hsp90.

FK506 binding to the 56-kilodalton immunophilin (Hsp56) in the glucocorticoid receptor heterocomplex has no effect on receptor folding or function.

It has recently been reported that the hsp56 component of glucocorticoid receptor heterocomplexes is an immunophilin of the FK506 binding class [Yem, A. W., Tomasselli, A. G., Heinrikson, R. L., Zurcher-Neely, H., Ruff, V. A., Johnson, R. A., & Deibel, M. R. (1992) J. Biol. Chem. 267, 2868-2871; Tai, P. K., Albers, M. W., Chang, H., Faber, L. E., & Schreiber, S. L. (1992) Science 256, 1315-1318]. The existence of binding proteins for these two potent groups of immunosuppressants in the same molecular complex compels us to ask whether FK506 affects glucocorticoid receptor function. We show here that hsp56 is a component of the native L-cell glucocorticoid receptor heterocomplex and that [3H]FK506 binds to the immunopurified, untransformed receptor complex. However, at concentrations in excess of those required to occupy all of its binding sites on hsp56, FK506 does not affect the steroid binding activity of the receptor nor does it stabilize or dissociate the receptor-hsp90 complex. FK506 does not affect steroid-mediated hsp90 dissociation from the receptor in vitro, and it does not affect steroid-mediated nuclear transfer of the receptor or steroid-mediated transcriptional enhancement from a reporter in intact cells. When immunopurified mouse glucocorticoid receptor is reconstituted into a heat shock protein complex by rabbit reticulocyte lysate, hsp56 is present in the reconstituted complex in addition to hsp90 and hsp70. FK506, however, does not affect reconstitution of the complex or return of the receptor to the steroid binding state, a change of conformation that occurs upon receptor association with hsp90.(ABSTRACT TRUNCATED AT 250 WORDS)

Steroid receptor folding by heat-shock proteins and composition of the receptor heterocomplex.

Over the past 2 years, reports from several laboratories have supported the proposal that the steroid receptors are bound through the hormone-binding domain to a protein complex that contains three heat-shock proteins-hsp90, hsp70, and hsp56. This receptor-heat-shock-protein heterocomplex accounts for the behavior of the classic 9 S, non-DNA-binding form of the adrenocorticoid, sex hormone, and dioxin receptors. The receptor heterocomplex has now been reconstituted by an enzymatic system in reticulocyte lysate. This represents the first in vitro system for reversing receptor transformation, and this ability to reconstitute the receptor heterocomplex promises rapid advances in our understanding of how these receptors are folded, transported, and regulated by hormone in the cell.

Energy-dependent conversion of transformed cytosolic glucocorticoid receptors from soluble to particulate-bound form.

We have recently published that soluble cytosolic glucocorticoid receptors are converted to a particulate form when they are incubated at 37 degrees C in a tubulin-polymerizing buffer [Pratt, W. B., Sanchez, E. R., Bresnick, E. H., Meshinchi, S., Scherrer, L. C., Dalman, F. C., & Welsh, M. J. (1989) Cancer Res. (Suppl.) 49, 2222s-2229s]. In this work, we further define this phenomenon and demonstrate that the L-cell glucocorticoid receptors are binding to a protein particulate composed largely of cytoskeletal proteins. Incubation of L-cell cytosol with glutamate at 37 degrees C converts the glucorticoid receptor to a form that pellets when cytosol is centrifuged at 150000g. The particulate material formed in a temperature-dependent and glutamate-dependent manner contains a large amount of tubulin, actin, and vimentin, but it is not the product of a cold-labile, colchicine-sensitive polymerization process. Very few cytosolic proteins are present in this complex, but the glucocorticoid receptor is tightly bound to it. Binding of the receptor to the cytoskeletal complex occurs after receptor transformation and is at least partially energy-dependent. Examination of the behavior of beta-galactosidase receptor fusion proteins and the nti glucocorticoid receptor demonstrates that residues 445 to the COOH-terminus of the receptor (DNA-binding and hormone-binding domains) contain the features required for binding to the cytoskeletal complex. Although it is the transformed receptor that associates tightly with the complex, DNA-binding activity is not required for association with the cytoskeletal particulate.(ABSTRACT TRUNCATED AT 250 WORDS)

A heat shock protein complex isolated from rabbit reticulocyte lysate can reconstitute a functional glucocorticoid receptor-Hsp90 complex.

When unliganded glucocorticoid receptor that has been stripped free of associated proteins is incubated with rabbit reticulocyte lysate, the receptor becomes associated with the 70- and 90-kDa heat shock proteins (hsp70 and hsp90), and the untransformed state of the receptor is functionally reconstituted [Scherrer, L. C., Dalman, F. C., Massa, E., Meshinchi, S., & Pratt, W. B. (1990) J. Biol. Chem. 265, 21397-21400]. Recently, an hsp70-containing protein complex (200-250 kDa) purified from rabbit reticulocyte lysate was shown to maintain a fusion protein bearing the mitochondrial matrix-targeting signal in a state that is competent for mitochondrial import [Sheffield, W. P., Shore, G. C., & Randall, S. K. (1990) J. Biol. Chem. 265, 11069-11076]. In this work, we show that this partially purified mitochondrial import-competent fraction contains both hsp90 and hsp70. When the purified fraction is immunoadsorbed with a monoclonal antibody specific for hsp90, a significant portion of the hsp70 is co-immunoadsorbed, suggesting that hsp90 and hsp70 are present together as a complex. The partially purified fraction maintains a hybrid precursor protein containing the mitochondrial matrix-targeting signal of rat pre-ornithine carbamyl transferase in an import-competent state. Incubation of immunopurified glucocorticoid receptor with this fraction of reticulocyte lysate results in ATP-dependent association of the receptor with both hsp70 and hsp90, and the resulting complexes are functional as assessed by return of the receptor to the high-affinity steroid binding conformation. The glucocorticoid receptor hetero-complex reconstituting activity of the lysate fraction is low relative to its mitochondrial import activity. Importantly, however, this is the first demonstration of the functional and structural reconstitution of the untransformed state of any steroid receptor utilizing a partially purified system.

Monovalent cation selectivity for ATP-dependent association of the glucocorticoid receptor with hsp70 and hsp90.

We have reported previously that incubation of the immunopurified transformed hormone-free glucocorticoid receptor with rabbit reticulocyte lysate reconstitutes the receptor complex with hsp90 and that reconstitution is accompanied by concomitant repression of DNA binding activity and regeneration of the steroid binding conformation (Scherrer, L. C., Dalman, F. C., Massa, E., Meshinchi, S., and Pratt, W. B. (1990) J. Biol. Chem. 265, 21397-21400). In this work we further characterize this system by defining the small M(r) components of reticulocyte lysate required for both structural and functional reconstitution of the receptor-hsp90 complex. Reconstitution is ATP-dependent and there is a direct relationship between the extent of hsp90 binding to the receptor and the number of specific steroid binding sites that are generated. Dialysis of reticulocyte lysate inactivates its reconstituting activity. Addition of an ATP-regenerating system or readdition of small M(r) lysate components (in the form of a Centricon C30 filtrate) has little effect, but the presence of both restores full reconstituting activity to dialyzed lysate, as assayed by steroid binding activity and by the binding of hsp90 and hsp70 to the receptor. The small M(r) activity is heat-stable, and it can be completely replaced by NH+4, K+, and Rb+, with K+ producing a maximal effect at the concentration normally present in undialyzed lysate. Na+ and Li+ have no reconstituting activity. This ion selectivity demonstrates that a monovalent cation binding site is involved in receptor heterocomplex reconstitution. It is intriguing that the protein unfoldase (e.g. clathrin uncoating ATPase) activity of hsp70 is known to have a similar monovalent cation dependence, and that under all conditions where hsp90 becomes bound to the receptor, we find that hsp70 is also bound.

Association of the transformed glucocorticoid receptor with a cytoskeletal protein complex.

In a recent paper we described a system in which glucocorticoid receptors associate with particulate complexes containing tubulin [Cancer Res. 49 (1989) 2222s-2229s]. When L cell cytosol is mixed with a microtubule stabilizing buffer and heated to 37 degrees C, the receptor becomes associated with a complex that can be centrifuged out of solution at 150,000 g. In this work we show that the glucocorticoid receptor-cytoskeletal protein complex forms in a temperature and glutamate-dependent manner. Molybdate does not affect generation of the cytoskeletal protein complex but it inhibits association of the receptor with the complex. This suggests that transformation of the receptor to its DNA-binding form is required for interaction with the cytoskeletal complex. Colchicine has no effect on generation of the particulate complex or on the association of receptor with it, suggesting that formation of the complex does not represent a classic in vitro process of tubulin polymerization.

A model of glucocorticoid receptor unfolding and stabilization by a heat shock protein complex.

It has recently been reported that incubation of avian progesterone receptors, mouse glucocorticoid receptors, or the viral tyrosine kinase pp60src with rabbit reticulocyte lysate reconstitutes their association with the 90 kDa heat shock protein, hsp90. The reassociation is thought to require unfolding of the steroid receptor or pp60src before hsp90 can bind. The unfoldase activity may be provided by hsp70, which is also present in the reconstituted receptor heterocomplex. In this paper we review evidence that hsp70 and hsp90 are associated in cytosolic heterocomplexes that contain a limited number of other proteins. From an analysis of known receptor-hsp interactions and a predicted direct interaction between hsp90 and hsp70 we have developed an admittedly very speculative model of glucocorticoid receptor unfolding and stabilization. One important feature of the model is that the receptor becomes attached to a heat shock protein heterocomplex rather than undergoing independent unfolding and stabilization events. The model requires that hsp70 and hsp90 bind directly to the receptor at independent sites. Importantly, the model accommodates the stoichiometry of 2 hsp90 per 1 molecule of receptor that has been assayed in the untransformed GR heterocomplex in cytosols prepared from hormone-free cells.

Localization of the 90-kDa heat shock protein-binding site within the hormone-binding domain of the glucocorticoid receptor by peptide competition.

In this work, we used two approaches to localize the 90-kDa heat shock protein (hsp90)-binding site within the hormone-binding domain of the glucocorticoid receptor. In the first approach, derivatives of the glucocorticoid receptor deleted for increasing portions of the COOH terminus were translated in rabbit reticulocyte lysate, and the [35S]methionine-labeled translation products were immunoadsorbed with the 8D3 monoclonal antibody against hsp90. The data suggest that a segment from amino acids 604 to 659 (mouse) of the receptor is required for hsp90 binding. We have recently shown that the internal deletion mutant of the mouse receptor (delta 574-632) binds hsp90, although the complex is somewhat unstable (Housley, P. R., Sanchez, E. R., Danielsen, M., Ringold, G. M., and Pratt, W. B. (1990) J. Biol. Chem. 265, 12778-12781). The two observations indicate that amino acids 574-659 are involved in forming a stable receptor-hsp90 complex and that region 632-659 is especially important. To test this hypothesis directly, we synthesized three peptides corresponding to segments in region 624-665 and three peptides spanning the highly conserved sequence at amino acids 582-617, and we then tested the ability of the peptides to compete for the association of hsp90 with the L cell glucocorticoid receptor. In this assay, the immunopurified hsp90-free mouse receptor is incubated with rabbit reticulocyte lysate, which directs the association of rabbit hsp90 with the mouse receptor, simultaneously converting the receptor to the steroid binding state. All three peptides spanning region 624-665 and a peptide corresponding to segment 587-606 inhibited both hsp90 association with the receptor and reconstitution of steroid binding capacity. The data from all of the approaches support a two-site model for the hsp90-binding site in which the critical contact site occurs in region 632-659, which contains a short proline-containing hydrophobic segment and adjacent dipole-plus-cysteine motif that are conserved among all of the hsp90-binding receptors in the superfamily. A second hsp90 contact site is predicted in region 574-632, which contains the only highly conserved amino acid sequence in the receptor superfamily outside of the DNA-binding domain.

Structural and functional reconstitution of the glucocorticoid receptor-hsp90 complex.

Untransformed steroid receptors in cytosol preparations are associated with the 90-kDa heat shock protein hsp90, but the study of how hsp90 affects receptor function has been held back by the inability to reassociate steroid receptors with hsp90 in cell-free systems. Recently we showed (Dalman, F.C., Bresnick, E. H., Patel, P. D., Perdew, G. H., Watson, S. J., and Pratt, W. B. (1989) J. Biol. Chem. 264, 19815-19821) that glucocorticoid receptors translated in rabbit reticulocyte lysate bind to hsp90 at the termination of receptor translation. In this work we show that rabbit reticulocyte lysate promotes the temperature-dependent association of hsp90 with immunopurified mouse L cell glucocorticoid receptors. Reticulocyte lysate also promotes the temperature-dependent dissociation of hormone-free receptors from a prebound receptor-DNA complex. The glucocorticoid receptor is released from DNA in association with rabbit hsp90, and reconstitution of the receptor-hsp90 complex is accompanied by complete restitution of steroid binding activity and repression of DNA binding activity. This is the first time that transformation of a DNA-bound steroid receptor has been reversed and it raises the question of whether the same or a similar system is involved in the termination of transcriptional activation when steroid dissociates from DNA-bound receptors in intact cells.

The relationship between glucocorticoid receptor binding to Hsp90 and receptor function.

In this minireview we summarize evidence that the association of the glucocorticoid receptor (GR) with hsp90 may determine three functional states of the receptor. First, there is a direct correlation between hsp90 binding to the receptor and repression of DNA binding activity. Temperature-dependent dissociation of hsp90 from the cytosolic GR-hsp90 complex is promoted by hormone with simultaneous conversion of the receptor to the DNA binding state. GR that is translated in rabbit reticulocyte lysate binds to hsp90 at or near the termination of receptor translation and is in the non-DNA-binding form. Second, there is a direct correlation between binding of the immunopurified GR to hsp90 and the presence of a high affinity steroid binding conformation of the receptor. GR translated in reticulocyte lysate binds steroid with high affinity, but GR translated in wheat germ extract is not bound to hsp90, does not bind steroid with high affinity, and is in the DNA-binding form. When immunopurified, hsp90-free GR is incubated with rabbit reticulocyte lysate, hsp90 associates with the receptor and high affinity steroid binding capacity is completely reactivated. Third, there is a correlation between binding of hsp90 to steroid receptors and their retention in an inactive "docking" state until the binding of hormone in the intact cell triggers a progression to high affinity nuclear binding sites where the primary events involved in transcriptional activation occur. In contrast to the receptors that are retained in the docking state, the unliganded thyroid hormone receptor proceeds directly to high affinity nuclear binding sites. Consistent with this difference in behavior, the thyroid hormone receptor is translated in reticulocyte lysate in its DNA binding form and is not associated with hsp90.

Hormone-free mouse glucocorticoid receptors overexpressed in Chinese hamster ovary cells are localized to the nucleus and are associated with both hsp70 and hsp90.

In this work, we examine the cellular localization and protein interactions of mouse glucocorticoid receptors that have been overexpressed in Chinese hamster ovary (CHO) cells (Hirst, M. A., Northrop, J. P., Danielsen, M., and Ringold, G. M. (1990) Mol. Endocrinol. 4, 162-170). We demonstrate that wild-type unliganded mouse glucocorticoid receptor, which is expressed in CHO cells to a level approximately 10 times that of L cells, is localized entirely to the nucleus by indirect immunofluorescence with the BuGR antireceptor monoclonal antibody. Overexpressed receptors that have either no hormone binding activity or no DNA binding activity because of point mutations also localize to the nucleus, providing genetic proof that the nuclear localization cannot reflect a steroid-mediated shift of the receptor from the cytoplasm to the nucleus and that DNA binding activity is not required for nuclear localization. Like unliganded progesterone receptors, which also associate in a loosely bound "docking" complex with the nucleus, the mouse glucocorticoid receptor overexpressed in CHO cells is associated with both hsp90 and hsp70. This is in contrast to the untransformed mouse glucocorticoid receptor in L cell cytosol, which is associated with hsp90 but not hsp70. The difference in hsp70 association between cell types could reflect overexpression of the receptor in CHO cells. However, like receptors in CHO cells selected for very high levels of overexpression, receptors in CHO cells selected for an intermediate level of receptor expression that is comparable to that of L cells are also bound to hsp70. This observation argues against an explanation of hsp70 association based purely on receptor overexpression, and we speculate that association of the unliganded glucocorticoid receptor with hsp70 might be a consequence of its nuclear localization in the CHO cells. Although there are differences between the mouse receptor in CHO cells and L cells, the nuclear localization signal of the untransformed mouse receptor reacts equivalently with the AP64 antibody against NL1 in cytosols prepared from both cell types.

Interaction of the glucocorticoid receptor with the Mr 90,000 heat shock protein: an evolving model of ligand-mediated receptor transformation and translocation.

Reports from several laboratories support a model for glucocorticoid receptor (GR) transformation in cytosol in which a heteromeric 9S complex of GR and the Mr 90,000 heat shock protein undergo a temperature-dependent and hormone-promoted dissociation to yield the free DNA-binding form of the receptor. In this paper, we review evidence that the 9S heteromeric complex is derived from the normal inactive state of the receptor in the intact cell and that both Mr 90,000 heat shock protein and the untransformed GR localize by immunofluorescence with specific monoclonal antibodies to microtubules in a variety of cell types in culture. We propose that an association with cytoskeleton may be required for translocating the GR from its cytoplasmic site of synthesis to its nuclear site of action and that the 9S complex is derived from this cytoskeleton-associated form. Similar molybdate-stabilized 9S complexes can be obtained for all of the steroid receptors, several of which clearly are localized to the nucleus prior to exposure to hormone. These receptors may have moved to the terminus of the translocation pathway where they remain in a cytoskeleton-bound "docking" position. We speculate that, in the intact cell, ligand-dependent dissociation of Mr 90,000 heat shock protein permits the steroid receptors to progress by some ordered mechanism to their high affinity sites of action within the nucleus.

Evidence that the 90-kilodalton heat shock protein is associated with tubulin-containing complexes in L cell cytosol and in intact PtK cells.

It has been established that the 90-kilodalton murine heat shock protein, hsp90, is associated with the untransformed, non-DNA-binding form of the glucocorticoid receptor in L cell cytosol. In this work, we show that incubation of L cell cytosol with Affi-Gel-coupled monoclonal antibodies directed against either alpha-tubulin alone or both alpha- and beta-tubulin results in the immune-specific adsorption of hsp90 identified by Western blotting with the AC88 monoclonal antibody. Similarly, the AC88 antibody, which is specific for hsp90, causes the immune-specific isolation of both alpha- and beta-tubulin from hypotonic cytosol. The distribution of hsp90 in cultured Potorous tridactylis kidney cells was examined by indirect immunofluorescence using the AC88 monoclonal as primary antibody. In interphase cells, AC88-dependent fluorescence was distributed like antitubulin antibody-dependent fluorescence in a fibrillar array located in the cytoplasm and around the periphery of the nucleus. In cells undergoing mitosis, AC88 fluorescence was located in the mitotic spindle. These observations suggest that a significant portion of hsp90 is associated with a tubulin-containing complex both in a hypotonic cytosol preparation from mouse fibroblasts and in intact marsupial kidney epithelial cells. The distribution of AC88 fluorescence in interphase Potorous tridactylis kidney cells is similar to the distribution of glucocorticoid receptor demonstrated by Wikstrom, A. C., Bakke, O., Okret, S., Bronnegard, M., and Gustafsson, J. A in rat hepatoma and human uterine cells.