A Cell Model for Conditional Profiling of Androgen-Receptor-Interacting Proteins

K A Mooslehner,I A Hughes,J D Davies

doi:10.1155/2012/381824

K A Mooslehner, I A Hughes + Show 1 more

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https://doi.org/10.1155/2012/381824

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Abstract

Partial androgen insensitivity syndrome (PAIS) is associated with impaired male genital development and can be transmitted through mutations in the androgen receptor (AR). The aim of this study is to develop a cell model suitable for studying the impact AR mutations might have on AR interacting proteins. For this purpose, male genital development relevant mouse cell lines were genetically modified to express a tagged version of wild-type AR, allowing copurification of multiprotein complexes under native conditions followed by mass spectrometry. We report 57 known wild-type AR-interacting proteins identified in cells grown under proliferating and 65 under nonproliferating conditions. Of those, 47 were common to both samples suggesting different AR protein complex components in proliferating and proliferation-inhibited cells from the mouse proximal caput epididymus. These preliminary results now allow future studies to focus on replacing wild-type AR with mutant AR to uncover differences in protein interactions caused by AR mutations involved in PAIS.

Highlights

Androgen insensitivity gives rise to a wide spectrum of disorders in man, the most severe being complete sex reversal, to milder forms of Partial androgen insensitivity syndrome (PAIS) associated with ambiguous or underdeveloped genitalia, or even milder forms causing “only” male infertility in otherwise healthy males
A second criteria was the nuclear localization of N-Terminal Tandem Affinity Purification Tag (N-TAP)-mAR, which was examined in several clones by immunofluorescence using FLAG and androgen receptor (AR)-specific antibodies
The transactivation properties of N-TAP-mAR were tested in COS cells by cotransfection of a GRE reporter construct, with expression levels confirmed by Western blot (Figures 2(b) and 2(c))

Summary

Introduction

Androgen insensitivity gives rise to a wide spectrum of disorders in man, the most severe being complete sex reversal, to milder forms of PAIS associated with ambiguous or underdeveloped genitalia, or even milder forms causing “only” male infertility in otherwise healthy males. Where no mutations are identified in the AR [2] mutations in AR coregulators may be implicated in failure to activate or repress androgen-regulated target genes. There are a number of mouse models available to study impaired AR function in vivo [3], the signalling networks are too complex to dissect without using simpler cell models. The aim of this study was to develop a cell model for the study of AR signalling in the urogenital tract. In turn this may identify disrupted signalling resulting from AR mutations associated with PAIS. By focussing solely on known AR coregulators, we were able (as a proof of principle) to uncover differences in the proteome of proliferating and nonproliferating epithelial PC1 cells

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