Background and Purpose: Melanocortin receptors (MC1–5) areinvolved in controlling appetite, glucocorticoid production, and modu-lating anti‐inflammatory responses. These functions are mediatedthrough binding with different melanocortin peptides. MC2expressionand activation was previously found to be regulated by twomelanocortin receptor accessory proteins, MRAP1 and MRAP2. ThePRESTO‐TANGO (parallel receptorome expression and screening viatranscriptional output, with transcriptional activation followingarrestin translocation) assay was designed to study in vitro G‐proteincoupled signalling. This assay reports MC receptor activation bylinking beta‐arrestin recruitment to the transcription of luciferasewhich generates light from catalysis of its substrate, luciferin. Theaim of this study was to examine the human MC3responsiveness tothe melanocortin ligands when co‐expressed withMRAP1using the PRESTO‐TANGO in vitro assay.
Experimental Approach:Modified HEK293 (HTLA) cells weretransfected using PolyJet™with 5 ng of MC3‐Tango plasmid withand without co‐transfected 5‐ng MRAP1‐pCDNA3.1‐FLAG. TotalDNA per well of a 96 plate was 50 ng (supplemented by pCDNA3.1).The transfected cells were stimulated 48‐hr post‐transfection witheither ACTH(1–39), NDP‐α‐MSH, or D‐Trp8‐γ3‐MSH (1 × 10−6–1×10−12M) for 20 hr. Luminescence was determined using a lumi-nescent plate reader (GloMax®) and log dose–response curves gener-ated withn=4–6.MC3‐Tango stimulation with and without MRAP1co‐transfection was compared using unpaired two‐tailedttest.Key Results:EC50values determined with the PRESTO‐Tango assayfor MC3were ACTH(1–39), 51.24 nM; NDP‐α‐MSH, 153.3 nM; andD‐Trp8‐γ3‐MSH, 78.32 nM. The co‐expression ofMC3withMRAP1led to a significant increase in the sensitivity of MC3to ACTH(1–39)(35.43 nM,P= .0099,n= 6); however, the effect of co‐expressingMC3and MRAP1 with NDP‐α‐MSH (204 nM,P= .5122,n= 4) andTrp8‐γ3‐MSH (77.6 nM,P= .4964,n= 4) was not significant.
TABLE 1ReceptorMRAP1co‐expression LigandEC50± SEM (nM)nMC3—ACTH(1–39)51.24 ± 2.056MC3YesACTH(1–39)35.43 ± 3.456MC3—NDP‐α;;‐MSH 153.3 ± 10.144MC3YesNDP‐α;;‐MSH 204 ± 77.984MC3—D‐Trp8‐γ3‐MSH 78.32 ± 1.724MC3YesD‐Trp8‐γ3‐MSH 77.6 ± 1.654Conclusions and Implications:This is the first report about the study-ing co‐expression of human MC3with MRAP1 in an in vitro assay.MRAP1 significantly increases the MC3affinity to ACTH(1–39)butnot to NDP‐α;;‐MSH or D‐Trp8‐γ3‐MSH, showing that melanocortinreceptor accessory proteins specifically alter melanocortin receptoractivation by different melanocortin ligands.