Endocrine Abstracts

The Melanocortin 2 Receptor (MC2R) is best known for its role in the adrenal cortex where it couples the action of ACTH to steroidogenesis, however it is also expressed by the adipocyte. We set out to characterize the up-regulation of the MC2R in the 3T3-L1 fibroblast, which can be induced to undergo adipogenesis.

RT-PCR using primers specific to exons 1 and 2 amplified three bands in differentiated 3T3-L1 adipocytes which sequencing revealed to be distinct MC2R splice variants. A 292bp amplicon was found to contain a novel 86bp exon. RT-PCR of individual splice variants demonstrated an up-regulation of mRNA 24hrs following hormonal induction but the newly described splice variant displayed a delayed up-regulation and was not detected until 48hrs.

Cells stably transfected with a promoter luciferase reporter were used to assay promoter activity over the time course of differentiation. These results demonstrate a 1.7 fold activation of the promoter after 24hrs corresponding with the appearance of mRNA transcripts. Promoter activity reached a peak of 3.3 fold on day 3 by which time all splice variants were maximally detectable.

A competitive binding assay for cAMP was used to map the response of differentiating cells to ACTH stimulation as a measure of the presence of functional receptor. Using this method, receptor activity was first observed on day two post induction when a 4.7 fold increase in cAMP was measured in stimulated cells. A maximal ACTH response on day 4 followed the peak in promoter activity on day 3.

RT-PCR demonstrates that the MC2R promoter is functioning in 3T3-L1 cells in the absence of Steroidogenic Factor-1 (SF-1), which has been shown to be necessary for transcription of the human and mouse MC2R in adrenal cells. We therefore propose that an SF-1 independent mechanism for MC2R transcription must be operating in the adipocyte.