Investigation of a melanocortin receptor:nucleoporin interaction that reveals a potentially novel aspect of ACTH action

Lead Research Organisation: Queen Mary, University of London
Department Name: Unlisted

Abstract

It is now well recognized that disturbances in the production and metabolism of the steroid hormone cortisol may have an important role in the development of many diseases. Cortisol production is mainly controlled by the pituitary hormone, ACTH. ACTH circulates in the blood and acts on a specific receptor in the adrenal gland to stimulate cortisol synthesis and secretion. Some aspects of the function of this receptor are not entirely clear and the possibility of additional signaling actions has been recognized for many years. Almost serendipitously we recently identified a protein known as Nup50 that interacts with the ACTH receptor. We found that stimulating adrenal cells with ACTH resulted in the receptor-Nup50 complex moving from the cell surface to the cell nucleus. Nup50 was previously identified as a protein that is intimately involved in the transport of proteins and other larger molecules into the nucleus. How this function relates to ACTH signaling will be the main focus of this project. Specifically we will investigate the role of the cell cycle and receptor internalization in Nup50 responses and we will use confocal microscopy to define where precisely in the cell the receptor-Nup50 complex resides. We will use molecular techniques to identify precisely which bits of each protein are involved in the interaction, and finally we will ask whether other related receptor proteins might also use this candidate signaling mechanism. This should provide us with a greatly enhanced understanding of this potential pathway, allowing us to assess its importance and relevance for human biology and disease.

Technical Summary

There is a significant body of data over many years suggesting the presence of certain G protein-coupled receptors (GPCRs) in the nucleus. In some cases, a functional role for these nuclear GPCRs has been identified. Furthermore, certain GPCR-associated proteins such as beta-arrestin-1 may translocate to the nucleus. In recent work, initiated in a search for ACTH receptor (melanocortin 2 receptor ? MC2R) chaperones and trafficking factors, we identified an interaction between the C-terminal tail of the MC2R and the nucleoporin, Nup50. This was confirmed using GST pull-down technology, and the receptor could readily co-immunoprecipitate Nup50 in H295R cells which express both proteins endogenously. Stimulation of these cells with ACTH induces translocation of this MC2R-Nup50 complex from the plasma membrane to the nucleus. These findings imply the presence of a novel action of ACTH in adrenal cells, the ultimate consequence of which remains uncertain. This project aims to characterise this phenomenon and we will (a) characterize the regulation of the MC2R-Nup50 complex formation and translocation in relation to the cell cycle and receptor internalization, examining the effect of cell cycle synchronization and internalization inhibitors. (b) Undertake detailed imaging of the MC2R, Nup50 and the complex in H295R cells using confocal immunocytochemistry and FRET imaging. (c) GST-pull down will be used to characterize the sites of the molecular interaction between the two components, and, depending on these findings, (d) the interaction of Nup50 with other melanocortin receptors will be investigated. Whilst these studies are likely to raise further questions, they should provide a strong basis for understanding this phenomenon and its potential significance.

Publications

10 25 50
 
Title MC2R BRET constructs 
Description Expression vectors with various tagged MC2R constructs 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact None that I am aware of as yet 
 
Description GPCR BRET analysis 
Organisation Semmelweiss University
Department Department of Physiology
Country Hungary 
Sector Academic/University 
PI Contribution USE of BRET in MC2R interactions
Collaborator Contribution Development of BRET techniques
Impact Cooray SN, Chung TT, Mazhar K, Szidonya L, Clark AJL. Bioluminescence Resonance Energy Transfer reveals the Adrenocorticotropin (ACTH)-induced conformational change of the activated ACTH receptor complex in living cells. Endocrinology (in press).
 
Description MRCT 
Organisation MRC-Technology
Department MRCT Centre for Therapeutics Discovery (CTD)
Country United Kingdom 
Sector Academic/University 
PI Contribution Information exchange, reagent supply and development of joint funding proposals (in progress and outcome awaited)
Collaborator Contribution Development and exploitation of BRET methods
Impact None as yet
Start Year 2010