Structure-function relationships of the mineralocorticoid receptor

Summary

The mineralocorticoid receptor (the receptor for the steroid hormone aldosterone) is an important therapeutic target in cardiovascular disease. We have identified interactions of the receptor that differ between the physiological ligands. Understanding these interactions and their structural basis may lead to the development of new therapeutic agents.

Description

The adrenal steroid, aldosterone, is a key regulator of blood pressure. Excessive aldosterone levels cause hypertension and contribute to the pathogenesis of cardiac disease. This laboratory has a program of work focused on various aspects of aldosterone action and the diseases that may arise from this action.

This project examines the receptor protein for aldosterone, the mineralocorticoid receptor (MR), which acts as a transcription factor to regulate gene expression.

We are examining several aspects of the function of the receptor using a range of molecular techniques including yeast-2 hybrid screening, mammalian-2-hybrid assays, transcription assays, mutagenesis and molecular modelling.

Currently, there are four areas of focus in this work.

Firstly, we are interested in understanding the way in which aldosterone or drugs that block the receptor bind. The crystal structure of the ligand-binding region of the receptor is known and we have used several approaches to analyse the functional aspects of this structure.

We are using a cross-species approach in which we are comparing the human MR with the zebra fish MR. The clinically used antagonist spironolactone, somewhat surprisingly, acts like aldosterone, as an agonist in the zebra fish ligand-binding domain. By creating chimeras between the zebra fish MR ligand-binding domain and the human MR ligand-binding domain, we hope to identify the amino acid differences (in what are otherwise two highly conserved molecules), that differentiate between a ligand being agonist and antagonist. This has implications for the future design of drugs to specifically and unambiguously block the MR.

Secondly, we have identified an interaction between two distinct domains in the full-length receptor, an interaction between the N-terminal domain and the ligand-binding domain which is at the C-terminus of the receptor (N/C-interaction). Such an interaction is also seen in the closely related androgen receptor, where it has a clear functional significance. In a series of now published studies, we have started the characterisation of this interaction in the MR.

It is of particular interest that, in the human, the interaction is blocked by the agonists cortisol deoxycorticosterone. This is an unexpected finding which is not observed when examining the interaction with the zebra fish MR. Again, the comparative biology approach will aid in dissecting the basis of the interaction. We are also in the process of identifying which region(s) in the N-terminus mediate the interaction. We are also establishing a FRET assay to study the interaction in the full-length receptor. Ultimately we hope to generate a transgenic mouse which has an MR lacking the N/C interaction.

Thirdly, the MR is able to signal through 3 principal mechanisms: the classic mechanism in which it binds to DNA and directly regulates gene expression; an interaction with other transcriptor factors which involves DNA but not the MR binding to the DNA transrepression; and a rapid action that occurs at the cell membrane which does not involve DNA.

We have proposed the generation of a transgenic mouse which lacks DNA binding in order to identify the relative contribution of these 3 signalling mechanisms to the actions of the mineralocorticoid receptor.

Fourthly, nuclear receptors after binding to the DNA, interact with the transcriptional apparatus to regulate gene expression through interactions with co-regulatory molecules.

We have recently used 2-hybrid screens to identify a series of co-regulatory molecules of the mineralocorticoid receptor which we are now in the process of characterising. It is of interest that a subset of these receptors is able to distinguish between the aldosterone-bound MR and the cortisol-bound MR. This is a novel finding and has potential implications for tissue-specific differences and thus for drug design.

Funding

National Health and Medical Research Council
National Heart Foundation

Outcomes

Identification of the basis of agonist binding and hence of the mechanisms by which better antagonists of the mineralocorticoid receptor might be designed.

Selected Publications

Pippal, J. B., Yao, Y., Rogerson, F. M. and Fuller, P.J. (2009) Structural and functional characterisation of the interdomain interaction in the mineralocorticoid receptor. Molecular Endocrinology (in press).

Pippal, J. B. and Fuller, P.J. (2008) Structure-function relationships in the mineralocorticoid receptor Journal of Molecular Endocrinology 41: 405-413.

Rogerson, F.M., Yao, Y., Elsass, R.E., Dimopoulos, N., Smith, B.J. and Fuller, P.J. (2007) A critical region in the mineralocorticoid receptor for aldosterone binding and activation by cortisol. Molecular Endocrinology 21:817-828.

Fuller, P.J. (2006) The aldosterone receptor - new insights? Expert Opinion in Investigational Drugs 15: 201-203.

Fuller, P.J., Smith, B.J. and Rogerson, F.M. (2004) Cortisol resistance in the New World revisited. Trends in Endocrinology and Metabolism 15: 296-299.

Rogerson, F.M., Yao, Y., Smith, B.J. and Fuller, P.J.(2004) Differences in the determinants of eplerenone, spironolactone and aldosterone binding to the mineralocorticoid receptor. Clinical and Experimental Physiology and Pharmacology 31: 704-710.

Rogerson, F.M., Brennan, F.E., and Fuller, P.J. (2004) Mineralocorticoid receptor binding, structure and function. Molecular and Cellular Endocrinology 217:203-212.

Rogerson, F.M., Dimopoulos, N., and Fuller, P.J. (2003) Interdomain interactions in the mineralocorticoid receptor. Molecular and Cellular Endocrinology 200: 45-55.

Rogerson, F.M., Brennan, F.E., and Fuller, P.J. (2003) Dissecting mineralocorticoid receptor structure and function. Journal of Steroid Biochemistry and Molecular Biology 85: 389-396.

Rogerson, F.M., Yao, Y-Z., Smith, B.J., Dimopoulos, N. and Fuller, P.J. (2003) Determinants of spironolactone binding specificity in the mineralocorticoid receptor. Journal of Molecular Endocrinology 31: 573-582.

Rogerson, F.M., Dimopoulos, N., Sluka, P., Chu, S., Curtis, A. J. and Fuller, P.J. (1999) Structural determinants of aldosterone binding selectivity in the mineralocorticoid receptor. Journal of Biological Chemistry 274: 36305-36311.