Identification of permissive insertion sites for generating functional fluorescent mineralocorticoid receptors.

Abstract:

:The mineralocorticoid receptor (MR), a member of the nuclear receptor superfamily of transcription factors, is activated by aldosterone and mediates its natriferic action in tight epithelia. MR is also expressed in nonepithelial tissues. Importantly, it mediates the deleterious effects of inappropriately high aldosterone levels in the heart, in which it induces the development of cardiac fibrosis. Antagonism of MR in humans is useful in the treatment of severe cardiac failure and some forms of hypertension. Despite the important pathophysiological and pharmacological role of this receptor, many important questions about its cellular biology and functional roles remain unanswered. A major challenge in the study of MR is the unavailability of fully functional fluorescent derivatives of the receptor. In this study we have created a library of MR mutants with insertions of the yellow fluorescent protein in various internal locations in the receptor using a random-insertion transposon-based technique. Screening of this library using a transactivation assay allowed us to identify several fluorescent constructs that retain functionality. Detailed characterization of one of these construct showed that it induces aldosterone-target genes such as the epithelial Na(+) channel subunits and the serum and glucocorticoid-induced kinase 1 at physiological concentrations of aldosterone to an equal extent than the wild-type receptor. Furthermore, aldosterone affinity, hormone-induced nuclear translocation, DNA binding and regulation of nongenomic pathways are all indistinguishable from the wild-type receptor. This new set of fluorescent MR derivatives provides a useful tool for studying the cell biology of the receptor.

journal_name

Endocrinology

journal_title

Endocrinology

authors

Aguilar-Sánchez C,Hernández-Díaz I,Lorenzo-Díaz F,Navarro JF,Hughes TE,Giraldez T,Alvarez de la Rosa D

doi

10.1210/en.2012-1210

subject

Has Abstract

pub_date

2012-07-01 00:00:00

pages

3517-25

issue

7

eissn

0013-7227

issn

1945-7170

pii

en.2012-1210

journal_volume

153

pub_type

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