The structurally related immunophilins cyclophilin 40 (CyP-40) and FKBP52 have been identified as components of the unactivated estrogen receptor. Both immunophilins have a similar molecular architecture that includes a C-terminal segment with a tetratricopeptide repeat (TPR) domain predicted to mediate protein interaction. hsp90 is a common cellular target for CyP-40 and FKBP52. Deletion mutants of CyP-40 fused to glutathione S-transferase were immobilized on glutathione-agarose and then used in a rapid hsp90 retention assay to define regions of the CyP-40 C terminus that are important for hsp90 binding. Our evidence suggests that the TPR domain is not sufficient for stable association of CyP-40 with hsp90 and requires the participation of flanking acidic and basic residues clustered at the Nand C-terminal ends, respectively. Both microdomains are characterized by ␣-helical structures with segregated hydrophobic and charged residues. Corresponding regions were identified in FKBP52. By preincubating myometrial cytosol with lysates containing bacterially expressed FKBP52, we have shown that FKBP52 competes with CyP-40 for hsp90 binding. Our results raise the possibility of a mutually exclusive association of CyP-40 and FKBP52 with hsp90. This would lead to separate immunophilin-hsp90-receptor complexes and place the estrogen receptor under the control of distinct immunophilin signaling pathways.The immunophilin components of the unactivated estrogen receptor, cyclophilin 40 (CyP-40), 1 and FKBP52, share significant sequence homology in their C-terminal regions (1) and represent separate classes of peptidylprolyl cis-trans-isomerases with binding specificities for the immunosuppressants cyclosporin A and FK506, respectively (2). These immunophilins display a similar structural organization of their functional domains characterized by an N-terminal region with overlapping isomerase and ligand binding domains and a conserved C-terminal segment that incorporates a 3-unit tetratricopeptide repeat (TPR) domain terminated by a potential site for calmodulin binding (1). We have previously speculated that the TPR domain may mediate the protein interaction properties of . This is consistent with evidence that similar repeat units in members of the TPR gene family are involved in functional association with target proteins (3).FKBP52 binds hsp90 within steroid receptor complexes (4) and also exists in association with hsp90 in the absence of receptor (5, 6). The interaction of FKBP52 with hsp90 has been studied extensively (7), and there is recent evidence that the TPR domain, localized in the C-terminal region of FKBP52, is fundamentally important for hsp90 binding (8). The structural similarity between CyP-40 and FKBP52 has led several groups to propose that the immunophilins may have a similar or perhaps competing role in cellular function (8 -10). In this regard, a recent report describes the association of human CyP-40 with hsp90 and provides evidence that the C-terminal, FKBP52-like domain determines this interaction ...

Structurally related tetratricopeptide repeat motifs in steroid receptor-associated immunophilins and the STI1 homolog, Hop, mediate the interaction with a common cellular target, hsp90. We have identified the binding domain in hsp90 for cyclophilin 40 (CyP40) using a two-hybrid system screen of a mouse cDNA library. All isolated clones encoded the intact carboxyl terminus of hsp90 and overlapped with a common region corresponding to amino acids 558 -724 of murine hsp84. The interaction was confirmed in vitro with bacterially expressed CyP40 and deletion mutants of hsp90␤ and was delineated further to a 124-residue COOH-terminal segment of hsp90. Deletion of the conserved MEEVD sequence at the extreme carboxyl terminus of hsp90 precludes interaction with CyP40, signifying an important role for this motif in hsp90 function. We show that CyP40 and Hop display similar interaction profiles with hsp90 truncation mutants and present evidence for the direct competition of Hop and FK506-binding protein 52 with CyP40 for binding to the hsp90 COOH-terminal region. Our results are consistent with a common tetratricopeptide repeat interaction site for Hop and steroid receptorassociated immunophilins within a discrete COOH-terminal domain of hsp90. This region of hsp90 mediates ATP-independent chaperone activity, overlaps the hsp90 dimerization domain, and includes structural elements important for steroid receptor interaction.Peptidyl-prolyl isomerases are cellular proteins that can mediate changes in protein conformation by catalyzing cis-trans isomerization about amino acid-proline peptide bonds (1-3). Immunophilins represent the predominant group within the rapidly growing peptidyl-prolyl isomerase protein family that includes the cyclophilins and FK506-binding proteins (FKBPs) 1 identified as cellular targets for the immunosuppressant drugs cyclosporin A and FK506, respectively (4, 5). An overlap of ligand binding and catalytic domains in immunophilins results in an inhibition of isomerase activity in response to immunosuppressant drug interaction (4, 5).Cyclophilin 40 (CyP40) was first isolated and identified in association with the unactivated estrogen receptor (6) and shares structural and sequence homology with FKBP52, previously described as a common component of aposteroid receptor complexes (7,8). A third mammalian immunophilin, FKBP51, with significant identity to CyP40 and FKBP52, has recently been identified in the progesterone receptor complex (9). The structural similarity among CyP40, FKBP51, and FKBP52 is characterized by an amino-terminal immunophilin-like domain and a conserved carboxyl-terminal tetratricopeptide repeat (TPR) domain that mediates protein interaction (6, 9 -12). Within steroid receptor complexes the immunophilins bind competitively to heat shock protein hsp90 to form distinct immunophilin-hsp90-receptor complexes (9,13,14). Conserved structural features that determine immunophilin interaction with an identical site in hsp90 include the TPR domain together with adjacent subregions located ...