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Bioluminescence Resonance Energy Transfer Reveals the Adrenocorticotropin (ACTH)-Induced Conformational Change of the Activated ACTH Receptor Complex in Living Cells

Cooray, SN; Chung, TT; Mazhar, K; Szidonya, L; Clark, AJ (2011) Bioluminescence Resonance Energy Transfer Reveals the Adrenocorticotropin (ACTH)-Induced Conformational Change of the Activated ACTH Receptor Complex in Living Cells. ENDOCRINOLOGY, 152 (2). 495 - 502. ISSN 0013-7227 https://doi.org/10.1210/en.2010-1053
SGUL Authors: Clark, Adrian John L

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Abstract

Bioluminescence resonance energy transfer analysis is used to study the interaction between melanocortin 2 receptor (MC2R) accessory protein and the MC2R and provides evidence for protein kinase A-dependent conformational changes in the receptor complex following receptor activation. The melanocortin 2 receptor (MC2R) accessory protein (MRAP) is a small single-transmembrane domain protein that plays a pivotal role in the function of the MC2R. The pituitary hormone, ACTH, acts via this receptor complex to stimulate adrenal steroidogenesis. Using both coimmunoprecipitation and bioluminescence resonance energy transfer (BRET), we show that the MC2R is constitutively homodimerized in cells. Furthermore, consistent with previous data, we also show that MRAP exists as an antiparallel homodimer. ACTH enhanced the BRET signal between MC2R homodimers as well as MC2R-MRAP heterodimers. However, ACTH did not enhance the physical interaction between these dimers as determined by coimmunoprecipitation. Real-time BRET analysis of the MRAP-MC2R interaction revealed two distinct phases of the ACTH-dependent BRET increase, an initial complex series of changes occurring over the first 2 min and a later persistent increase in BRET signal. The slower ACTH-dependent phase was inhibited by the protein kinase A inhibitor KT5720, suggesting that signal transduction was a prerequisite for this later conformational change. The MRAP-MC2R BRET approach provides a unique tool with which to analyze the activation of this receptor.

Item Type: Article
Additional Information: This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/us/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Keywords: Adrenocorticotropic Hormone, Blotting, Western, Cell Line, Cyclic AMP, Humans, Immunoprecipitation, Protein Binding, Protein Conformation, Receptors, Corticotropin, Science & Technology, Life Sciences & Biomedicine, Endocrinology & Metabolism, ENDOCRINOLOGY & METABOLISM, ACCESSORY PROTEIN MRAP, MELANOCORTIN-2 RECEPTOR, HORMONE RECEPTOR, DIMERIZATION, TRAFFICKING, HOMODIMERS, OLIGOMERIZATION, HETERODIMERS, MECHANISM, STATES, 07 Agricultural And Veterinary Sciences, 11 Medical And Health Sciences, 06 Biological Sciences
Journal or Publication Title: ENDOCRINOLOGY
ISSN: 0013-7227
Dates:
DateEvent
1 February 2011Published
Web of Science ID: WOS:000286422100020
Download EPMC Full text (HTML)
URI: http://openaccess.sgul.ac.uk/id/eprint/101295
Publisher's version: https://doi.org/10.1210/en.2010-1053

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