Mutation of Hip's carboxy-terminal region inhibits a transitional stage of progesterone receptor assembly.

Steroid receptor complexes are assembled through an ordered, multistep pathway involving multiple components of the cytoplasmic chaperone machinery. Two of these components are Hsp70-binding proteins, Hip and Hop, that have some limited homology in their C-terminal regions, outside the sequences mapped for Hsp70 binding. Within this region of Hip is a DPEV sequence that occurs twice; in Hop, one DPEV sequence plus a partial second sequence occurs. In an effort to better understand Hip function as it relates to assembly of progesterone receptor complexes, the DPEV region of Hip was targeted for mutations. Each DPEV sequence was mutated to an APAV sequence, singly or in combination. The combined mutation, APAV2, was further combined with a deletion of Hip's tetratricopeptide repeat region that is required for Hsp70 binding or with a deletion of Hip's GGMP repeat. An additional mutant was prepared by truncation of Hip's DPEV-containing C terminus. By comparing interactions of various Hip forms with Hsp70, it was determined that mutation of the DPEV sequences created a dominant inhibitory form of Hip. The mutant Hip-Hsp70 complex was not prevented from interacting with progesterone receptor, but the mutant caused a dose-dependent inhibition of receptor assembly with Hsp90. The behavior of the Hip mutant is consistent with a model in which Hip and Hop are required to facilitate the transition from an early receptor complex with Hsp70 into later complexes containing Hsp90.

Molecular cloning of human FKBP51 and comparisons of immunophilin interactions with Hsp90 and progesterone receptor.

A cDNA for human FKBP51 has been cloned and sequenced, and protein products have been expressed in both in vitro and bacterial systems. The deduced amino acid sequence for human FKBP51 is 90% identical to sequences of recently described murine proteins and is 55% identical to the sequence of human FKBP52. Human FKBP51 mRNA is expressed in a wide range of tissues, and the protein has peptidylprolyl isomerase activity that is inhibited by FK506 but not cyclosporine. FKBP51 is the same as a previously described progesterone receptor-associated immunophilin that, similar to FKBP52 and cyclophilin 40, is an Hsp90-binding protein and appears in functionally mature steroid receptor complexes along with Hsp90 and p23. Each of the three receptor-associated immunophilins displays interactions with progesterone receptor that are more dynamic than Hsp90-receptor interactions. Whereas FKBP52 and FKBP51 compete about equally well for binding to Hsp90 in a purified system, FKBP51 accumulates preferentially in progesterone receptor complexes assembled in a cell-free system. This observation provides a precedent for differential interactions between Hsp90-associated immunophilins and target proteins such as steroid receptors.

Mutational analysis of the hsp70-interacting protein Hip.

The hsp70-interacting protein Hip participates in the assembly pathway for progesterone receptor complexes. During assembly, Hip appears at early assembly stages in a transient manner that parallels hsp70 interactions. In this study, a cDNA for human Hip was used to develop various mutant Hip forms in the initial mapping of functions to particular Hip structural elements. Hip regions targeted for deletion and/or truncation included the C-terminal region (which has some limited homology with Saccharomyces cerevisiae Sti1 and its vertebrate homolog p60), a glycine-glycine-methionine-proline (GGMP) tandem repeat, and a tetratricopeptide repeat (TPR). Binding of Hip to hsp70's ATPase domain was lost with deletions from the TPR and from an adjoining highly charged region; correspondingly, these Hip mutant forms were not recovered in receptor complexes. Truncation of Hip's Sti1-related C terminus resulted in Hip binding to hsp70 in a manner suggestive of a misfolded peptide substrate; this hsp70 binding was localized to the GGMP tandem repeat. Mutants lacking either the C terminus or the GGMP tandem repeat were still recovered in receptor complexes. Truncations from Hip's N terminus resulted in an apparent loss of Hip homo-oligomerization, but these mutants retained association with hsp70 and were recovered in receptor complexes. This mutational analysis indicates that Hip's TPR is required for binding of Hip with hsp70's ATPase domain. In addition, some data suggest that hsp70's peptide-binding domain may alternately or concomitantly bind to Hip's GGMP repeat in a manner regulated by Sti1-related sequences.

Interactions of p60, a mediator of progesterone receptor assembly, with heat shock proteins hsp90 and hsp70.

Previous studies on the assembly of progesterone receptor (PR) complexes in vitro have suggested that PR assembly is a dynamic, ordered process involving at least eight nonreceptor proteins. One of these proteins, p60, appears transiently during assembly and is not a component of functionally mature PR complexes. In the present study we observe that a monoclonal antibody specific for p60 can, on the one hand, inhibit formation of mature PR complexes containing heat shock protein 90 (hsp90), p23, and immunophilins and, on the other, enhance recovery of early PR complexes containing hsp70 and Hip (p48). This observation supports a model in which p60 functions at an intermediate stage of PR assembly to facilitate formation of subsequent PR complexes lacking p60. Since p60 is typically found in a complex with hsp90 and hsp70, we have further characterized its interactions with these proteins. P60 can bind either hsp70 or hsp90 independently and in an ATP-independent manner. Since hsp90 and hsp70 do not readily associate on their own, it appears that p60 is the central organizing component of an hsp90-p60-hsp70 complex. Mutational analysis of p60 indicates that the N terminus is required for hsp70 binding, and a central region containing tetratricopeptide repeat motifs is necessary for binding hsp90 and hsp70. The hsp90-p60-hsp70 multichaperone complex is highly dynamic and does not appear to be affected by the hsp90-binding drug geldanamycin. The interactions of hsp70 and hsp90 in intermediate PR complexes are shown to be distinct from their separate interactions in early PR complexes (hsp70) or in mature PR complexes (hsp90). From these results, it appears that p60 is a key mediator in the chaperoned assembly and functional maturation of PR complexes.

Molecular cloning of human p48, a transient component of progesterone receptor complexes and an Hsp70-binding protein.

A 48-kDa protein (p48) that transiently associates with progesterone receptor during cell-free assembly in rabbit reticulocyte lysate was isolated by two-dimensional gel separation. Tryptic peptide sequences were generated and used to develop an antipeptide antiserum recognizing p48 by Western immunostaining, and this antiserum was used to monitor purification of native p48 from reticulocyte lysate. Eight mouse monoclonal antibodies capable of immunoprecipitating vertebrate p48 were generated. These monoclonal antibodies served as probes to clone ten p48 cDNAs from a HeLa cDNA expression library. One of the cloned cDNAs was sequenced in its entirety and codes for a 369-amino acid protein (calculated Mr = 41,324). Northern blot analysis of RNA from multiple human tissues suggest that p48 may be ubiquitously expressed. Expression of human p48 cDNA in vitro yielded a product that comigrated with rabbit p48 by SDS-PAGE and associated with progesterone receptor in a similar manner. Immunoprecipitation of p48 complexes revealed a common association of p48 with hsp70 and, to a lesser extent, with hsp90 and p60. Thus, it appears that p48 is a novel component of the cytoplasmic molecular chaperone machinery.

Progesterone receptor structure and function altered by geldanamycin, an hsp90-binding agent.

The assembly of progesterone receptor (PR) heterocomplexes in vitro involves at least eight components of the molecular chaperone machinery, and as earlier reports have shown, these proteins exhibit complex, dynamic, but ordered, interactions with one another and PR. Using the selective hsp90 binding agent geldanamycin (GA), we have found that PR assembly in vitro can be arrested at a previously observed intermediate assembly step. Like mature PR complexes, the intermediate complexes contain hsp90, but they differ from mature complexes by the presence of hsp70, p60, and p48 and the absence of immunophilins and p23. Arrest of PR assembly is likely due to GA's ability to directly block binding of p23 to hsp90. An important functional consequence of GA-mediated assembly arrest in vitro is the inability of the resulting PR complexes to bind progesterone, despite the presence of hsp90 in the receptor complexes. The biological significance of the in vitro observations is demonstrated by GA's ability to (i) rapidly block PR's hormone binding capacity in intact cells and (ii) alter the composition of COS cell PR complexes in a manner similar to that observed during in vitro reconstitutions. An updated model for the cyclic assembly pathway of PR complexes that incorporates the present findings with earlier results is presented.

Identification of serotonin 5HT2 receptors in bovine pineal gland.

The concentration of serotonin in the pineal gland is extremely high, which prompted speculation that in addition to serving as a precursor of melatonin, serotonin may have an independent function of its own. By using [3H]-spiperone as a ligand, and ketanserine as a selective serotonin 5HT2 receptor antagonist, we have identified 5HT2 receptor in the bovine pineal gland, revealing a single population of binding sites with a dissociation equilibrium constant (Kd) value of 1.26 +/- 0.41 nM and a receptor density (Bmax) value of 193 +/- 38.85 fmol/mg protein. In displacement experiments, the concentrations of the drugs required to inhibit 50% of the specific binding of [3H]-spiperone in descending order of potency were methysergide greater than ritanserin greater than pirenperone greater than pipamperone greater than ketanserin greater than cyproheptadine greater than M-trifluoromethylphenyl-piperazine greater than prazosin greater than 5-methoxy-N-N-dimethyltryptamine hydrogen oxalate greater than 1-(3-chlorophenol) piperazine greater than serotonin. In the rat pineal gland, [3H]-spiperone revealed a low affinity serotonin binding site with a Kd value of 25.77 +/- 10.7 nM and a Bmax value of 1244 +/- 472 fmol/mg protein. The results of these studies are interpreted to indicate that the bovine pineal gland possess serotonin 5HT2 receptor. However, the rat pineal gland possess a serotoninergic binding site of unknown nature.