ADAMTS-9 in Mouse Cartilage Has Aggrecanase Activity That Is Distinct from ADAMTS-4 and ADAMTS-5.

A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 are the principal aggrecanases in mice and humans; however, mice lacking the catalytic domain of both enzymes (TS-4/5∆cat) have no skeletal phenotype, suggesting there is an alternative aggrecanase for modulating normal growth and development in these mice. We previously identified aggrecanase activity that (a) cleaved at E↓G rather than E↓A bonds in the aggrecan core protein, and (b) was upregulated by retinoic acid but not IL-1α. The present study aimed to identify the alternative aggrecanase. Femoral head cartilage explants from TS-4/5∆cat mice were stimulated with IL-1α or retinoic acid and total RNA was analysed by microarray. In addition to ADAMTS-5 and matrix metalloproteinase (MMP)-13, which are not candidates for the novel aggrecanase, the microarray analyses identified MMP-11, calpain-5 and ADAMTS-9 as candidate aggrecanases upregulated by retinoic acid. When calpain-5 and MMP-11 failed to meet subsequent criteria, ADAMTS-9 emerged as the most likely candidate for the novel aggrecanase. Immunohistochemistry revealed ADAMTS-9 expression throughout the mouse growth plate and strong expression, particularly in the proliferative zone of the TS-4/5-∆cat mice. In conclusion, ADAMTS-9 has a novel specificity for aggrecan, cleaving primarily at E↓G rather than E↓A bonds in mouse cartilage. ADAMTS-9 might have more important roles in normal skeletal development compared with ADAMTS-4 and ADAMTS-5, which have key roles in joint pathology.

Effects of stimulated aggrecanolysis on nanoscale morphological and mechanical properties of wild-type and aggrecanase-resistant mutant mice cartilages.

A key event in arthritis pathogenesis is the degradation of aggrecan, the major component in articular cartilage. In this work, we investigate the effects of stimulated aggrecanolysis on the morphological and nanomechanical properties of cartilage harvested from wild-type mice and aggrecanase-resistant mutant mice named "Jaffa". The cartilages were native or were subjected to stimulated aggrecanolysis by interleukin-1[Formula: see text] (IL-1[Formula: see text]) treatment. The nanoscale morphological and mechanical properties of the sectioned cartilages were measured by using a sharp probe by atomic force microscopy (AFM). The IL-1[Formula: see text] treatment resulted in a higher nanoroughess and stiffness of the cartilage from wild-type mice. However, the same treatment did not lead to any measurable change in the nanoroughness or stiffness of the cartilage from mutant mice Jaffa. This suggests that blocking aggrecanolysis by genetic modification has created the stability in the structures and mechanical properties of the cartilage at nanoscale. The present study provides insight into the mechanism of aggrecan degradation, which can complement the examination by biochemical and histological techniques.

A Disintegrin and Metalloproteinase with Thrombospondin Motifs-5 (ADAMTS-5) Forms Catalytically Active Oligomers.

The metalloproteinase ADAMTS-5 (A disintegrin and metalloproteinase with thrombospondin motifs) degrades aggrecan, a proteoglycan essential for cartilage structure and function. ADAMTS-5 is the major aggrecanase in mouse cartilage, and is also likely to be the major aggrecanase in humans. ADAMTS-5 is a multidomain enzyme, but the function of the C-terminal ancillary domains is poorly understood. We show that mutant ADAMTS-5 lacking the catalytic domain, but with a full suite of ancillary domains inhibits wild type ADAMTS activity, in vitro and in vivo, in a dominant-negative manner. The data suggest that mutant ADAMTS-5 binds to wild type ADAMTS-5; thus we tested the hypothesis that ADAMTS-5 associates to form oligomers. Co-elution, competition, and in situ PLA experiments using full-length and truncated recombinant ADAMTS-5 confirmed that ADAMTS-5 molecules interact, and showed that the catalytic and disintegrin-like domains support these intermolecular interactions. Cross-linking experiments revealed that recombinant ADAMTS-5 formed large, reduction-sensitive oligomers with a nominal molecular mass of ∼ 400 kDa. The oligomers were unimolecular and proteolytically active. ADAMTS-5 truncates comprising the disintegrin and/or catalytic domains were able to competitively block full-length ADAMTS-5-mediated aggrecan cleavage, measured by production of the G1-EGE(373) neoepitope. These results show that ADAMTS-5 oligomerization is required for full aggrecanase activity, and they provide evidence that blocking oligomerization inhibits ADAMTS-5 activity. The data identify the surface provided by the catalytic and disintegrin-like domains of ADAMTS-5 as a legitimate target for the design of aggrecanase inhibitors.

Identification and characterization of a ligand-selective mineralocorticoid receptor coactivator.

The mineralocorticoid receptor (MR) is unique in responding to 2 physiological ligands: aldosterone and cortisol. In epithelial tissues, aldosterone selectivity is determined by the activity of 11ß-hydroxysteroid dehydrogenase type 2. In other tissues, cortisol is the primary ligand. To understand the structural determinants of ligand-specific MR activation, we sought to identify coregulatory molecules that interact with the ligand-binding domain (LBD) of the MR. A yeast-2-hybrid (Y2H) kidney cDNA library was screened with the human MR-LBD in the presence of aldosterone and cortisol. One clone, identified as aldosterone-specific in the Y2H assay, exhibited a 7-fold greater response, aldosterone vs. cortisol, in a mammalian-2-hybrid (M2H) assay. This clone encodes the region of the tesmin gene that has 2 leucine-x-x-leucine-leucine (LxxLL) motifs. Full-length tesmin coactivates (>2-fold) MR-mediated transactivation in the presence of aldosterone, but not of cortisol; this specificity is observed with a range of promoters. GST pulldown and coimmunoprecipitation of the MR by tesmin supports a direct interaction, mediated by the 2 LxxLL motifs. Tesmin thus represents a novel MR coregulator that exhibits a differential interaction, providing further evidence of the adoption of ligand-dependent conformations by the MR-LBD.

Investigating ADAMTS-mediated aggrecanolysis in mouse cartilage.

Proteolysis of the cartilage proteoglycan aggrecan is a feature of arthritis. We present a method for analyzing aggrecanolysis in in vitro cultures of 3-week-old mouse femoral head cartilage based on traditional methods developed for large animal species. Investigators can choose either a simple analysis that detects several aggrecan fragments released into culture medium only or a more comprehensive study that detects all fragments present in both the medium and the cartilage matrix. The protocol comprises (i) cartilage culture and optional cartilage extraction, (ii) a quick and simple colorimetric assay for quantitating aggrecan and (iii) neoepitope western blotting to identify specific aggrecan fragments partitioning to the medium or cartilage compartments. The crucial difference between the methods for mice and larger animals is that the proportion of aggrecan in a given sample is normalized to total aggrecan rather than to tissue wet weight. This necessary break from tradition arises because tiny volumes of liquid clinging to mouse cartilage can increase the apparent tissue wet weight, causing unacceptable errors. The protocol has broad application for the in vitro analysis of transgenic mice, particularly those with mutations that affect cartilage remodeling, arthritic disease and skeletal development. The protocol is robust, reliable and takes 7-11 d to complete.

Cytokine-induced increases in ADAMTS-4 messenger RNA expression do not lead to increased aggrecanase activity in ADAMTS-5-deficient mice.

OBJECTIVE: To compare the regulation of aggrecanase messenger RNA (mRNA) and enzyme activity by proinflammatory cytokines in primary mouse chondrocytes. METHODS: Primary chondrocytes were isolated from knee epiphyses of 6-8-day-old mice and cultured as monolayers. The cells were incubated with tumor necrosis factor α (TNFα), oncostatin M (OSM), or interleukin-6 (IL-6)/soluble IL-6 receptor, and mRNA levels were measured by quantitative polymerase chain reaction at various time points. To measure aggrecanase activity, the cells were incubated with cytokine in the presence of exogenous aggrecan, and substrate cleavage was measured using antibodies to neoepitopes. RESULTS: Expression of both ADAMTS-4 and ADAMTS-5 mRNA was up-regulated by TNFα and OSM. ADAMTS-5 mRNA expression was also up-regulated by IL-6. Treatment of wild-type mouse chondrocytes with each of the 3 cytokines increased cleavage of aggrecan at Glu(373)↓(374) Ala and Glu(1670)↓(1671) Gly; in chondrocytes lacking ADAMTS-5 activity, there was negligible cleavage at either site despite increased expression of ADAMTS-4 mRNA in the presence of TNFα or OSM. None of the cytokines substantially altered mRNA expression of ADAMTS-1 or ADAMTS-9. CONCLUSION: Despite substantial increases in the expression of ADAMTS-4 mRNA induced by TNFα and OSM, these cytokines induced little if any increase in aggrecanolysis in ADAMTS-5-deficient mouse chondrocytes. Our data show a poor correlation between the level of cytokine-induced ADAMTS-4 mRNA expression and the level of aggrecan-degrading activity in cultured chondrocytes.

Structural and functional characterization of the interdomain interaction in the mineralocorticoid receptor.

The mineralocorticoid receptor (MR) plays a central role in electrolyte homeostasis and in cardiovascular disease. We have previously reported a ligand-dependent N/C-interaction in the MR. In the present study we sought to fully characterize the MR N/C-interaction. By using a range of natural and synthetic MR ligands in a mammalian two-hybrid assay we demonstrate that in contrast to aldosterone, which strongly induces the interaction, the physiological ligands deoxycorticosterone and cortisol weakly promote the interaction but predominantly inhibit the aldosterone-mediated N/C-interaction. Similarly, progesterone and dexamethasone antagonize the interaction. In contrast, the synthetic agonist 9alpha-fludrocortisol robustly induces the interaction. The ability of the N/C interaction to discriminate between MR agonists suggests a subtle conformational difference in the ligand-binding domain induced by these agonists. We also demonstrate that the N/C interaction is not cell specific, consistent with the evidence from a glutathione-S-transferase pull-down assay, of a direct protein-protein interaction between the N- and C-terminal domains of the MR. Examination of a panel of deletions in the N terminus suggests that several regions may be critical to the N/C-interaction. These studies have identified functional differences between physiological MR ligands, which suggest that the ligand-specific dependence of the N/C-interaction may contribute to the differential activation of the MR that has been reported in vivo.

Evidence of a novel aggrecan-degrading activity in cartilage: Studies of mice deficient in both ADAMTS-4 and ADAMTS-5.

OBJECTIVE: To characterize aggrecan catabolism and the overall phenotype in mice deficient in both ADAMTS-4 and ADAMTS-5 (TS-4/TS-5 Delta-cat) activity. METHODS: Femoral head cartilage from the joints of TS-4/TS-5 Delta-cat mice and wild-type mice were cultured in vitro, and aggrecan catabolism was stimulated with either interleukin-1alpha (IL-1alpha) or retinoic acid. Total aggrecan release was measured, and aggrecanase activity was examined by Western blotting using neoepitope antibodies for detecting cleavage at EGE 373-374 ALG, SELE 1279-1280 GRG, FREEE 1467-1468 GLG, and AQE 1572-1573 AGEG. Aggrecan catabolism in vivo was examined by Western blotting of cartilage that had been extracted immediately ex vivo. RESULTS: TS-4/TS-5 Delta-cat mice were viable, fertile, and phenotypically normal. TS-4/TS-5 Delta-cat cartilage explants did not release aggrecan in response to IL-1alpha, and there was no detectable increase in aggrecanase neoepitopes. TS-4/TS-5 Delta-cat cartilage explants released aggrecan in response to retinoic acid. There was no retinoic acid-stimulated cleavage at either EGE 373-374 ALG or AQE 1572-1573 AGEG. There was a low level of cleavage at SELE 1279-1280 GRG and major cleavage at FREEE 1467-1468 GLG. Ex vivo, cleavage at FREEE 1467-1468 GLG was substantially reduced, but still present, in TS-4/TS-5 Delta-cat mouse cartilage compared with wild-type mouse cartilage. CONCLUSION: An aggrecanase other than ADAMTS-4 and ADAMTS-5 is expressed in mouse cartilage and is up-regulated by retinoic acid but not IL-1alpha. The novel aggrecanase appears to have different substrate specificity from either ADAMTS-4 or ADAMTS-5, cleaving E-G bonds but not E-A bonds. Neither ADAMTS-4 nor ADAMTS-5 is required for normal skeletal development or aggrecan turnover in cartilage.

Distinguishing aggrecan loss from aggrecan proteolysis in ADAMTS-4 and ADAMTS-5 single and double deficient mice.

Aggrecan loss from mouse cartilage is predominantly because of ADAMTS-5 activity; however, the relative contribution of other proteolytic and nonproteolytic processes to this loss is not clear. This is the first study to compare aggrecan loss with aggrecan processing in mice with single and double deletions of ADAMTS-4 and -5 activity (Deltacat). Cartilage explants harvested from single and double ADAMTS-4 and -5 Deltacat mice were cultured with or without interleukin (IL)-1alpha or retinoic acid and analyzed for (i) the kinetics of (35)S-labeled aggrecan loss, (ii) the pattern of (35)S-labeled aggrecan fragments released into the media and retained in the matrix, (iii) the pattern of total aggrecan fragments released into the media and retained in the matrix, and (iv) specific cleavage sites within the interglobular and chondroitin sulfate-2 domains. The loss of radiolabeled aggrecan from ADAMTS-4/-5 Deltacat cartilage was less than that from ADAMTS-4, ADAMTS-5, or wild-type cartilage under nonstimulated conditions. IL-1alpha and retinoic acid stimulated radiolabeled aggrecan loss from wild-type and ADAMTS-4 Deltacat cartilage, but there was little effect on ADAMTS-5 cartilage. Proteolysis of aggrecan contributed most to its loss in wild-type, ADAMTS-4, and ADAMTS-5 Deltacat cartilage explants. The pattern of proteolytic processing of aggrecan in these cultures was consistent with that occurring in cartilage pathologies. Retinoic acid, but not IL-1alpha, stimulated radiolabeled aggrecan loss from ADAMTS-4/-5 Deltacat cartilage explants. Even though there was a 300% increase in aggrecan loss from ADAMTS-4/-5 Deltacat cartilage stimulated with retinoic acid, the loss was not associated with aggrecanase cleavage but with the release of predominantly intact aggrecan consistent with the phenotype of the ADAMTS-4/-5 Deltacat mouse. Our results show that chondrocytes have additional mechanism for the turnover of aggrecan and that when proteolytic mechanisms are blocked by ablation of aggrecanase activity, nonproteolytic mechanisms compensate to maintain cartilage homeostasis.

A critical region in the mineralocorticoid receptor for aldosterone binding and activation by cortisol: evidence for a common mechanism governing ligand binding specificity in steroid hormone receptors.

The amino acids that confer aldosterone binding specificity to the mineralocorticoid receptor (MR) remain to be determined. We had previously analyzed a panel of chimeras created between the MR and the glucocorticoid receptor and determined that amino acids 804-874 of the MR ligand binding domain are critical for aldosterone binding. In the present study a further series of chimeras was created within this region. The chimeras were analyzed by a transactivation assay and [(3)H]aldosterone binding, and the critical region was narrowed down to amino acids 820-844. Site-directed mutagenesis was used to create single and multiple amino acid substitutions in this region. These studies identified 12 of the 16 amino acids that differ in the MR and the glucocorticoid receptor in this region as being critical to conferring aldosterone responsivity. The amino acids that differ in the region 820-844 lie on the surface of the molecule and, therefore, it appears that MR ligand binding selectivity is conferred by residues that do not form part of the ligand binding pocket. Other studies have found that the corresponding regions of the androgen and glucocorticoid receptors are critical for the binding of natural and synthetic ligands, suggesting a common mechanism governing ligand binding specificity. The new chimeras also displayed, as previously reported, a dissociation between cortisol binding and transactivation and, intriguingly, only those that bound aldosterone with high affinity were activated by cortisol, suggesting a common mechanism that underlies specificity of aldosterone binding and the ability of cortisol to activate the MR.

ADAMTS-5 deficiency does not block aggrecanolysis at preferred cleavage sites in the chondroitin sulfate-rich region of aggrecan.

In the mouse, proteolysis in the aggrecan interglobular domain is driven by ADAMTS-5, and mice deficient in ADAMTS-5 catalytic activity are protected against aggrecan loss and cartilage damage in experimental models of arthritis. Here we show that despite ablation of ADAMTS-5 activity, aggrecanolysis can still occur at two preferred sites in the chondroitin sulfate-rich region. Retinoic acid was more effective than interleukin-1alpha (IL) in promoting cleavage at these sites in ADAMTS-5-deficient cartilage. These results suggest that cleavage at preferred sites in the chondroitin sulfate-rich region is mediated by ADAMTS-4 or an aggrecanase other than ADAMTS-5. Following retinoic acid or IL-1alpha stimulation of cartilage explants, aggrecan fragments in medium and extracts contained SELE(1279) or FREEE(1467) C-terminal sequences. Some SELE(1279) and FREEE(1467) fragments were retained in the cartilage, with intact G1 domains. Other SELE(1279) fragments were released into the medium and co-migrated with the (374)ALGS neoepitope, indicating they were aggrecanase-derived fragments. In contrast none of the FREEE(1467) fragments released into the medium co-migrated with the (374)ALGS neoepitope, suggesting that, despite their size, these fragments were not products of aggrecanase cleavage in the interglobular domain. ADAMTS-5, but not ADAMTS-1, -4, or -9, was up-regulated 8-fold by retinoic acid and 17-fold by IL-1alpha treatment. The data show that whereas ADAMTS-5 is entirely responsible for cleavage in the interglobular domain, cleavage in the chondroitin sulfate-rich region is driven either by ADAMTS-4, which compensates for loss of ADAMTS-5 in this experimental system, or possibly by another aggrecanase. The data show that there are differential aggrecanase activities with preferences for separate regions of the core protein.

ADAMTS-1-knockout mice do not exhibit abnormalities in aggrecan turnover in vitro or in vivo.

OBJECTIVE: To determine the role of the proteinase ADAMTS-1 in normal and accelerated catabolism of aggrecan in articular and growth plate cartilage of mice. METHODS: Expression of ADAMTS-1 was determined using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of RNA isolated from microdissected chondrocytes from different zones of mouse growth plate and articular cartilage. Real-time RT-PCR for ADAMTS-4, ADAMTS-5, and ADAMTS-9 was performed on femoral head cartilage of wild-type (WT) and ADAMTS-1-knockout (KO) mice. Histologic and immunohistologic evaluation of growth plate and articular cartilage was performed in WT and KO mice from birth to 12 weeks of age. The effect of ADAMTS-1 ablation on cartilage proteoglycan loss was studied in antigen-induced arthritis (AIA). Aggrecan catabolism in WT and KO mice was studied in an in vitro model of cartilage degradation, by quantitation of glycosaminoglycan loss and histologic, immunohistologic, and Western immunoblot analyses. RESULTS: ADAMTS-1 messenger RNA (mRNA) was expressed in normal mouse articular and growth plate cartilage and was up-regulated in terminal hypertrophic differentiation of growth plate chondrocytes. There was no difference in mRNA levels in the cartilage of WT compared with KO mice for the other potential aggrecanases ADAMTS-4, ADAMTS-5, or ADAMTS-9. ADAMTS-1-KO mice were significantly smaller than their WT littermates; however, no morphologic differences between the genotypes were evident in growth plate or articular cartilage from birth to skeletal maturity (12-16 weeks). Similarly, no difference in cartilage aggrecan content or presence of aggrecan degradation products was detected between WT and KO mice. There was no difference between WT and KO mice in the degree of synovial inflammation or depletion of cartilage aggrecan in AIA. There was no difference between WT and KO cartilage in either basal or stimulated aggrecan loss in vitro; however, subtle changes in the aggrecanase-generated aggrecan catabolites were observed in interleukin-1-treated cartilage. CONCLUSION: Although ADAMTS-1 is expressed in articular and growth plate cartilage and is able to cleave aggrecan at physiologically relevant sites, our results indicate that it does not play a significant nonredundant role in normal cartilage and bone development and growth. Similarly, ablation of ADAMTS-1 offered no protection from accelerated aggrecanolysis in an inflammatory model of arthritis or in an in vitro model of early cartilage degradation. ADAMTS-1 does not appear to be a viable target for treatment of cartilage destruction in arthritis.

ADAMTS5 is the major aggrecanase in mouse cartilage in vivo and in vitro.

Aggrecan is the major proteoglycan in cartilage, endowing this tissue with the unique capacity to bear load and resist compression. In arthritic cartilage, aggrecan is degraded by one or more 'aggrecanases' from the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteinases. ADAMTS1, 8 and 9 have weak aggrecan-degrading activity. However, they are not thought to be the primary aggrecanases because ADAMTS1 null mice are not protected from experimental arthritis, and cleavage by ADAMTS8 and 9 is highly inefficient. Although ADAMTS4 and 5 are expressed in joint tissues, and are known to be efficient aggrecanases in vitro, the exact contribution of these two enzymes to cartilage pathology is unknown. Here we show that ADAMTS5 is the major aggrecanase in mouse cartilage, both in vitro and in a mouse model of inflammatory arthritis. Our data suggest that ADAMTS5 may be a suitable target for the development of new drugs designed to inhibit cartilage destruction in arthritis, although further work will be required to determine whether ADAMTS5 is also the major aggrecanase in human arthritis.

Differences in the determinants of eplerenone, spironolactone and aldosterone binding to the mineralocorticoid receptor.

The importance of mineralocorticoid receptor (MR) antagonists in the treatment of cardiovascular disease has been emphasised by two recent clinical trials, one using spironolactone and the other using a new selective MR antagonist, namely eplerenone. Eplerenone has a very low affinity for the glucocorticoid receptor (GR). Determinants of binding specificity of eplerenone to the MR were investigated using chimeras created between the ligand-binding domains (LBD) of the MR and the GR. These chimeras had been used previously to investigate aldosterone and spironolactone binding specificity to the MR. Eplerenone competed strongly for [(3)H]-dexamethasone binding to a MR/GR chimera containing amino acids 804-874 of the MR and weakly to a chimera containing amino acids 672-803 of the MR. Within the 804-874 region, eplerenone competed for [(3)H]-dexamethasone binding to a chimera containing amino acids 820-844 of the MR, although the calculated affinity was approximately 10-fold lower than for binding to the full-length MR LBD. Similar results were obtained using another MR antagonist, namely spironolactone. Modelling of eplerenone binding to the MR LBD, based on the GR LBD crystal structure, suggests that amino acids 820-844 affect the overall shape of the ligand-binding pocket and that eplerenone acts as an MR antagonist because it fails to stabilize the active conformation of the receptor. In contrast with results with the MR antagonists eplerenone and spironolactone, amino acids 820-844 are sufficient in themselves to confer high-affinity aldosterone binding to the MR, suggesting that the binding determinants of the two antagonists are similar to each other but differ from those of aldosterone.

Cortisol resistance in the New World revisited.

Insights into the molecular basis of glucocorticoid action have been obtained from the analysis of cortisol resistance. The glucocorticoid receptor (GR) in both New World primates and guinea pigs has a decreased affinity, in vivo, for cortisol; this is achieved by two distinct mechanisms. In the New World primates recent studies have identified a key role for co-chaperones. The amino acids responsible for cortisol resistance in the guinea pig GR lie not in the ligand-binding pocket but on the surface of the receptor. We hypothesize that this region might be the site of interaction between the co-chaperones and the GR, and hence that the resistance occurs through the same mechanism, albeit from opposite sides.

Mineralocorticoid receptor binding, structure and function.

The isolation of aldosterone 50 years ago was a critical first step in elucidating the mechanism by which corticosteroids regulate electrolyte homeostasis. The broad principles of this mechanism involving an intracellular receptor acting on specific genes to induce the expression/repression of aldosterone-induced proteins (AIP) were established 30 years ago. The cloning of the mineralocorticoid receptor (MR) has enabled studies of the subcellular mechanisms of aldosterone action, including the molecular dissection of structure-function relationships in the receptor. We have exploited the close structural and functional similarity of the MR with the glucocorticoid receptor to identify the regions in the MR that confer ligand-binding specificity. The critical region is located, not as might be expected in the ligand-binding pocket but rather on the surface of the molecule. These studies have been extended to an analysis of the interactions between the N-terminal and ligand-binding domains of the MR. In the last decade, AIP have been identified; the regulation of the genes encoding these AIP are discussed.

Dissecting mineralocorticoid receptor structure and function.

The molecular mechanisms by which aldosterone regulates epithelial sodium transport in the distal colon and the distal nephron remain to be fully elucidated. Aldosterone acts via the mineralocorticoid receptor (MR) to induce the expression of genes whose products are involved in sodium transport. The structural basis of MR interactions with aldosterone has been examined by creating chimeras of the MR and the closely related glucocorticoid receptor; we have exploited differences in ligand-binding specificity to determine the region(s) of the MR that confer aldosterone-binding specificity. These findings have been related to a three-dimensional model of the MR based on the crystal structure of the progesterone receptor. These studies have been extended to include the characterisation of interactions between the N- and C-termini of the MR. We have characterised six genes that are regulated in vivo in the distal colon and/or kidney of the rat that are directly and acutely regulated by aldosterone administration: the three subunits of the epithelial sodium channel, serum and glucocorticoid-induced kinase, channel-inducing factor and K-ras2A. These studies provide insights into the molecular pathways that mediate aldosterone-induced amiloride-sensitive epithelial sodium transport.

Interdomain interactions in the mineralocorticoid receptor.

The potential for interaction between the N-terminal domain and the C-terminal region (hinge and ligand-binding domain) of the mineralocorticoid receptor (MR) was examined using the mammalian-2-hybrid assay. The MR C-terminal region was fused to the GAL4 DNA-binding domain (GAL4-MRC). To examine if the AF-2 is involved in the interaction, as has been reported for other steroid hormone receptors, it was inactivated by point mutation (E962A). The N-terminal domain was fused to the VP16 transactivation domain (VP16-MRNT). In the mammalian-2-hybrid assay both GAL4-MRC and GAL4-MRC(E962A) interact with VP16-MRNT in an aldosterone-dependent manner. The GAL4-MRC(E962A) construct was used in subsequent experiments to examine the AF-2-independent N/C-interaction. The MR antagonist spironolactone inhibits the aldosterone-mediated association of the two domains. GAL4-MRC(E962A) interacts weakly with the GR or AR N-terminal domains in the presence of aldosterone. No dimerization between GAL4-MRC(E962A) and VP16-MRC is observed. Interestingly, cortisol produces a much weaker N/C-interaction than aldosterone, and it is possible that the N/C-interaction may contribute to observed functional differences in the MR bound to the two ligands.