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Ligand-induced conformational change in the human mineralocorticoid receptor occurs within its hetero-oligomeric structure.

ABSTRACT: To determine the first steps involved in the mechanism of action of aldosterone and its antagonists, we analysed the ligand-induced structural changes of the human mineralocorticoid receptor (hMR) translated in vitro. Limited chymotrypsin digestion of the receptor generated a 30 kDa fragment. Following binding of a ligand to hMR, the 30 kDa fragment became resistant to chymotrypsin proteolysis, indicating a change in the receptor conformation. Differences in sensitivity to chymotrypsin of the 30 kDa fragment were observed after binding of agonists and antagonists to hMR, suggesting that these two classes of ligands induced different hMR conformations. Several lines of evidence allowed us to identify the 30 kDa fragment as the subregion encompassing the C-terminal part of the hinge region and the ligand-binding domain (LBD) or hMR (hMR 711-984). (1) The 30 kDa fragment is not recognized by FD4, an antibody directed against the N-terminal region of hMR. (2) Aldosterone remains associated with the 30 kDa fragment after chymotrypsin proteolysis of the aldosterone-hMR complex. (3) A truncated hMR, lacking the last 40 C-terminal amino acids (hMR 1-944), yields a 26 kDa proteolytic fragment. In addition, we showed that the unbound and the aldosterone-bound 30 kDa fragment were both associated with heat-shock protein (hsp) 90, indicating that the ligand-induced conformational change takes place within the hetero-oligomeric structure and that the 711-984 region is sufficient for hsp90-MR interaction. We conclude that the ligand-induced conformational change of the receptor is a crucial step in mineralocorticoid action. It occurs within the LBD, precedes the release of hsp90 from the receptor and is dependent upon the agonist/antagonist nature of the ligand.

SUBMITTER: Couette B

PROVIDER: S-EPMC1217212 | biostudies-other | 1996 Apr

REPOSITORIES: biostudies-other

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Project description:Recently, we provided evidence that α1-adrenergic receptors (ARs) in vascular smooth muscle are regulated by chemokine (C-X-C motif) receptor (CXCR) 4 and atypical chemokine receptor 3 (ACKR3). While we showed that CXCR4 controls α1-ARs through formation of heteromeric receptor complexes in human vascular smooth muscle cells (hVSMCs), the molecular basis underlying cross-talk between ACKR3 and α1-ARs is unknown. We show that ACKR3 agonists inhibit inositol trisphosphate production in hVSMCs on stimulation with phenylephrine. In proximity ligation assays and co-immunoprecipitation experiments, we observed that recombinant and endogenous ACKR3 form heteromeric complexes with α1A/B/D-AR. While small interfering RNA knockdown of ACKR3 in hVSMCs reduced α1B/D-AR:ACKR3, CXCR4:ACKR3, and α1B/D-AR:CXCR4 complexes, small interfering RNA knockdown of CXCR4 reduced α1B/D-AR:ACKR3 heteromers. Phenylephrine-induced inositol trisphosphate production from hVSMCs was abolished after ACKR3 and CXCR4 small interfering RNA knockdown. Peptide analogs of transmembrane domains 2/4/7 of ACKR3 showed differential effects on heteromerization between ACKR3, α1A/B/D-AR, and CXCR4. While the transmembrane domain 2 peptide interfered with α1B/D-AR:ACKR3 and CXCR4:ACKR3 heteromerization, it increased heteromerization between CXCR4 and α1A/B-AR. The transmembrane domain 2 peptide inhibited ACKR3 but did not affect α1b-AR in β-arrestin recruitment assays. Furthermore, the transmembrane domain 2 peptide inhibited phenylephrine-induced inositol trisphosphate production in hVSMCs and attenuated phenylephrine-induced constriction of mesenteric arteries. α1-ARs form hetero-oligomeric complexes with the ACKR3:CXCR4 heteromer, which is required for α1B/D-AR function, and activation of ACKR3 negatively regulates α1-ARs. G protein-coupled receptor hetero-oligomerization is a dynamic process, which depends on the relative abundance of available receptor partners. Endogenous α1-ARs function within a network of hetero-oligomeric receptor complexes.

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Ligand-induced conformational change in the human mineralocorticoid receptor occurs within its hetero-oligomeric structure.

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