ISSN 1470-3947 (print)
ISSN 1479-6848 (online)

Searchable abstracts of presentations at key conferences in endocrinology

Published by BioScientifica
Endocrine Abstracts (2005) 9 OC27 
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Interaction of the ACTH receptor C-terminal tail with a nuclear envelope protein

M Doufexis & AJL Clark

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Adrenocorticotrophic hormone (ACTH), a 39 amino acid peptide is the principal regulator of adrenal steroidogenesis and is thus essential for life. ACTH mediates its effects through a cell surface G-protein coupled receptor which elevates the intracellular production of cAMP through adenylate cyclase. There have been significant difficulties in developing a suitable heterologous expression system to study the ACTH receptor (or melanocortin 2 receptor - MC2R), and we have postulated the existence of a specific factor or factors that are involved post-translationaly in the correct folding and/or trafficking of the receptor. We have used the bacterial two-hybrid system (B2H) to investigate intracellular interacting partners of the MC2R by cloning its C-terminal tail into the bait plasmid of the B2H system. This was used to screen a mouse adrenocortical Y6 cell cDNA library. Three independent screens were performed and led to the isolation on two separate occasions of the same cDNA fragment of 1.4 kb size which encoded a nuclear envelope protein of approximately 60 kd. This result was further investigated using a GST pull down assay. The C-terminal tail of the MC2R was fused in frame to the GST protein and the resultant fusion protein was used to confirm the interaction with the nuclear envelope protein. This is the first time a member of the melanocortin receptor family has been shown to interact in vitro with a nuclear envelope protein. However the endothelin ETB receptor has been shown to co-localise with a different nuclear envelope protein, Nup62, and to co-purify with Nup62 in nuclear fractions from a series of cells. These results lend support to the possibility of an additional intracellular function performed by the MC2R that might involve processes directly affecting gene regulation or nuclear membrane integrity.

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