Purification and characterization of recombinant human mineralocorticoid receptor.

Published

Journal Article

The mineralocorticoid receptor (MR) plays a critical role in the maintenance of electrolyte homeostasis and blood pressure via direct effects on the distal nephron and the cardiovascular system. The MR also has an important role in the pathology of cardiovascular disease, particularly heart failure, and is therefore an attractive therapeutic target. However, renal side effects limit its use in the clinic. Previous studies of MR molecular pharmacology have been performed on its isolated ligand-binding domain (LBD); however, current evidence suggests that nuclear receptor LBDs behave differently in isolation, than in the context of the full-length receptor. To date, technical issues have precluded production of full-length MR, thereby preventing molecular and structural studies of the MR LBD in its natural context. Here, we describe expression and purification of full-length human MR (hMR). hMR was expressed in Sf9 insect cells with an N-terminal biotinylated (bt)-tag, and stabilised by addition of ligand. bt-hMR exhibited ligand-binding and transactivation properties similar to that of the native protein. Affinity purification using an avidin matrix yielded approximately 120mug MR protein from 0.5lt Sf9 culture, and the receptor was purified bound to either aldosterone or cortisol. Recombinant hMR had a molecular weight of 110-130kDa, bound an MR DNA response element in vitro and interacted with a known co-regulator, PGC-1alpha, in GST pull-down assays, indicating its functional activity. Availability of this reagent will now enable analysis of MR structure and ligand interactions in the context of the full-length receptor, a prerequisite for future development of ligand-selective MR antagonists for the treatment of cardiovascular disease.

Full Text

Duke Authors

Cited Authors

  • Clyne, CD; Chang, C-Y; Safi, R; Fuller, PJ; McDonnell, DP; Young, MJ

Published Date

  • April 10, 2009

Published In

Volume / Issue

  • 302 / 1

Start / End Page

  • 81 - 85

PubMed ID

  • 19114086

Pubmed Central ID

  • 19114086

International Standard Serial Number (ISSN)

  • 0303-7207

Digital Object Identifier (DOI)

  • 10.1016/j.mce.2008.11.030

Language

  • eng

Conference Location

  • Ireland