Ulrich Gehring

Mammalian protein RAP46: an interaction partner and modulator of 70 kDa heat shock proteins

A ubiquitously expressed nuclear receptor‐associating protein of ∼46 kDa (RAP46) was identified recently. Interaction experiments with in vitro‐translated proteins and proteins contained in cell extracts revealed that a great variety of cellular regulators associate with RAP46. However, in direct interaction tests by the far‐Western technique, only 70 kDa proteins showed up and were identified as members of the 70 kDa heat shock protein (hsp70) family. Interaction is specific since not all members of the hsp70 family bind to RAP46; interaction occurs through their ATP‐binding domain. RAP46 forms complexes with hsp70 in mammalian cells and interacts with hsp70 in the yeast two‐hybrid system. Consistent with the fact that hsp70 can bind a multitude of proteins, we identified heteromeric complexes of RAP46–hsp70 with some selected proteins, most notably c‐Jun. Complex formation is increased significantly by pre‐treatment with alkaline phosphatase, thus suggesting modulation of interactions by protein phosphorylation. We observed that RAP46 interferes with efficient refolding of thermally denatured luciferase. Moreover, ATP‐dependent binding of misfolded proteins to hsp70 was greatly inhibited by RAP46. These data suggest that RAP46 functions as a regulator of hsp70 in higher eukaryotes.

The Function of Steroid Hormone Receptors Is Inhibited by the hsp90-specific Compound Geldanamycin

The ansamycin antibiotic geldanamycin, which specifically interacts with the heat shock protein hsp90, was used to study the function of hsp90 in steroid hormone receptors. We observed inhibition of glucocorticoid-specific gene induction in several responsive cell systems. Hormone binding abilities of receptors for glucocorticoid, progestin, androgen, and estrogen were inhibited upon exposing intact cells to geldanamycin. Inhibition was only seen when geldanamycin was applied to cell cultures under growth conditions or was present during in vitro synthesis; presynthesized receptors in cell extracts were not affected. Upon withdrawal of geldanamycin, glucocorticoid binding ability was regained; this was partially independent of de novo protein synthesis. Geldanamycin caused decreased levels of immunoreactive glucocorticoid receptors in wild-type cells with enhanced degradation occurring through the ubiquitin-proteasome pathway. Analysis of receptors from treated cells revealed a heteromeric structure of normal size in which the receptor polypeptide is complexed with normal amounts of hsp90 and the immunophilin p59. These data support the view that hsp90 actively participates in steroid-induced signal transduction, and they suggest that geldanamycin affects receptor action without disrupting hsp90-containing heterocomplexes per se. Nevertheless, complexes synthesized and assembled in vitro in the presence of geldanamycin differ from receptors of cellular origin.

RAP46 Is a Negative Regulator of Glucocorticoid Receptor Action and Hormone-induced Apoptosis

RAP46 was first identified by its ability to bind the glucocorticoid receptor. It has since been reported to bind several cellular proteins, including the anti-apoptotic protein Bcl-2, but the biological significance of these interactions is unknown. Here we show that RAP46 binds the hinge region of the glucocorticoid receptor and inhibits DNA binding and transactivation by the receptor. We further show that overexpression of RAP46 in mouse thymoma S49.1 cells inhibits glucocorticoid-induced apoptosis. Conversely, glucocorticoid-induced apoptosis and transactivation were enhanced after treating S49.1 cells with the immunosuppressant rapamycin, which down-regulates cellular levels of BAG-1, the mouse homolog of RAP46. The effect of rapamycin can, however, be overcome by overexpression of RAP46. These results together identify RAP46 as a protein that controls glucocorticoid-induced apoptosis through its negative regulatory action on the transactivation property of the glucocorticoid receptor.

Proteins interacting with the molecular chaperone hsp70/hsc70: physical associations and effects on refolding activity

We investigated several hsp70/hsc70 interacting proteins and established by two independent techniques that hsp40 and Hop/p60 specifically interact with the 257 residue carboxy‐terminal domain of hsp70 while Hap‐46 and Hip/p48 bind the 383 residue amino‐terminal ATP binding domain. Hap‐46 and Hip/p48 competed for binding to hsc70, while Hap‐46 had no effect on the binding of either Hop/p60 or hsp40 to hsc70. Hap‐46 inhibited the refolding of thermally denatured firefly luciferase in an hsc70 and hsp40 dependent assay, and this effect was largely compensated by Hop/p60. These interacting proteins thus appear to cooperate in affecting the chaperoning activity of hsp70/hsc70.

Chromosome assignment of a murine glucocorticoid receptor gene (Grl-1) using intraspecies somatic cell hybrids.

Abstract Hybrids between two mouse lymphoma cell lines, the glucocorticoid sensitive S49.1 and the resistant EL4 line, are sensitive to the cytolytic steroid effect as long as they retain the S49.1 specific glucocorticoid receptor and the complete chromosome complements contributed by both parental lines. With the use of the semisynthetic glucocorticoid dexamethasone, resistant segregants (dex R ) have been isolated which have lost the S49.1 specific receptor but retain the EL4 type of receptor. On the average these segregants have also lost one chromosome. We have carried out a detailed karyotype analysis of both parental cell lines and their hybrids using trypsin-Giemsa banding for chromosome identification. S49.1 cells contain 40 apparently normal chromosomes with monosomy X and trisomy 1. EL4 cells have 39 chromosomes, half of which are structurally abnormal. In addition to low levels of random karyotypic variability in S49.1 × EL4 hybrids we have observed one consistent difference between sensitive (dex s ) and dex R sublines: sensitive hybrids contained three chromosomes 18 while resistant segregants only had two. We were able to distinguish both chromosomes 18 contributed by the S49.1 parent and the one from the EL4 cell line by structural variations involving the centromeric heterochromatin and the nucleolar organizer regions. A specific chromosome 18 derived from S49.1 was consistently absent in 3 dex R segregant lines derived from different dex s hybrid clones. Since the dex R segregant hybrids have lost the S49.1 specific glucocorticoid receptor we conclude that the gene for the receptor is located on that chromosome 18 which is consistently lost. The other S49.1 derived 18 presumably carries a mutant or silent receptor gene, as S49.1 has previously been shown to have hemizygous levels of the receptor. This study demonstrates the usefulness of intraspecies somatic cell hybrids for mapping purposes. The overall karyotypic stability of such hybrids allows the identification of specific chromosomes eliminated by selection pressure.

Cellular receptor levels and glucocorticoid responsiveness of lymphoma cells

A series of mouse lymphoma cell lines of independent origin was investigated with respect to glucocorticoid sensitivity, cellular receptor levels, and properties of receptors. The concentrations of the glucocorticoid dexamethasone required to produce comparable growth-inhibitory effects varied considerably amongst these cell lines. Also a wide range in the number of receptors per cell was found. When the receptor-steroid complexes were compared with respect to nuclear binding properties and affinities for DNA, no differences were seen. For 7 out of 10 cell lines studied we obtained a direct correlation between hormonal sensitivity and the number of cellular receptor sites divided by the dissociation constants KD for the receptor-dexamethasone complexes. This suggests that the receptor is a major quantitative determinant for steroid responsiveness. The limitations of receptor measurements for glucocorticoid therapy of lymphoid neoplastic disease are discussed.

Heteromeric nature of glucocorticoid receptor

The wild‐type and a mutant receptor of S49.1 lymphoma cells have been shown by photoaffinity labelling to contain steroid‐binding polypeptides of M r 94 000 and 40 000, respectively. We investigated the molybdate‐stabilized forms of these receptors and obtained M r 325 000 and 285 000, respectively, by gel filtration and sedimentation analysis. Mild chymotrypsin treatment of the large wild‐type receptor resulted in a form of about M r 290 000 which contained a steroid‐binding polypeptide of M r 40 000. The data suggest that the high‐M r forms of glucocorticoid receptors are heteromeric in nature and contain one steroid‐binding polypeptide per complex

Interference between Proteins Hap46 and Hop/p60, Which Bind to Different Domains of the Molecular Chaperone hsp70/hsc70

ABSTRACT Several structurally divergent proteins associate with molecular chaperones of the 70-kDa heat shock protein (hsp70) family and modulate their activities. We investigated the cofactors Hap46 and Hop/p60 and the effects of their binding to mammalian hsp70 and the cognate form hsc70. Hap46 associates with the amino-terminal ATP binding domain and stimulates ATP binding two- to threefold but inhibits binding of misfolded protein substrate to hsc70 and reactivation of thermally denatured luciferase in an hsc70-dependent refolding system. By contrast, Hop/p60 interacts with a portion of the carboxy-terminal domain of hsp70s, which is distinct from that involved in the binding of misfolded proteins. Thus, Hop/p60 and substrate proteins can form ternary complexes with hsc70. Hop/p60 exerts no effect on ATP and substrate binding but nevertheless interferes with protein refolding. Even though there is no direct interaction between these accessory proteins, Hap46 inhibits the binding of Hop/p60 to hsc70 but Hop/p60 does not inhibit the binding of Hap46 to hsc70. As judged from respective deletions, the amino-terminal portions of Hap46 and Hop/p60 are involved in this interference. These data suggest steric hindrance between Hap46 and Hop/p60 during interaction with distantly located binding sites on hsp70s. Thus, not only do the major domains of hsp70 chaperones communicate with each other, but cofactors interacting with these domains affect each other as well.

Tetrameric structure of the nonactivated glucocorticoid receptor in cell extracts and intact cells

Mouse lymphoma cells contain a nonactivated glucocorticoid receptor of M r∼330000 which is heteromeric in nature and is unable to bind to DNA. Following affinity labeling of the steroid‐binding subunit and subsequent cross‐linking with dimethyl suberimidate at various times either in cell extracts or in intact cells, a series of labeled bands was detected in SDS gels. From the molecular masses of completely and partially cross‐linked complexes we conclude that the large nonactivated receptor is a tetramer composed of two 90 kDa subunits, one 50 kDa polypeptide and one steroid‐binding subunit.

Steroid Hormone Receptors and Heat Shock Proteins

Publisher Summary This chapter describes the steroid hormone receptors, both in the unliganded and in the liganded states that are subject to phosphorylation on various serine, threonine, and tyrosine residues. The fact that nonactivated steroid hormone receptors are of much higher molecular weights than the respective receptor polypeptides evoked the possibility of homotetrameric structures as is the case in many multimeric proteins. The heteromeric forms of steroid hormone receptors are able to bind the hormone but do not interact with DNA. Thus, receptor-associated proteins, most notably hsp90, appear to play a dual role in modulating receptor properties. Geldanamycin may affect hsp90-associated protein kinases in a different manner. In several instances, the disruption of complexes with hsp90 proteins has been observed, leading to impaired protein kinase signaling. Even though not all the details of receptor assembly with heat shock proteins are clear, the observations with the hsp90-specific agent geldanamycin further support the chaperoning role of hsp90 complexes in receptor assembly.