Identification of the Translation Start Site of the Human Melanocortin 3 Receptor

Abstract

Background: The melanocortin-3-receptor (MC3R) is a G-protein coupled receptor participating in hypothalamic energy metabolism. So far, it was assumed that the translation of the human MC3R starts at the non-conserved first ATG, however, a second evolutionary conserved ATG is located 37 amino acids downstream. One frequent polymorphism, T6K, is located between these two ATGs. Methods: For characterization of the two potential start ATGs, COS-7 cells were transfected with plasmids encoding the longer and the shorter form of the human MC3R. For signal transduction properties, cAMP was measured. Cell surface expression was determined by using an ELISA method. The translational start point of the MC3R was investigated by a GFP-based method. Results: Signal transduction was comparable for the long and the short receptor form. Cell surface expression via aminoterminal hemagglutinin tag could only be detected in the shorter form, but not in the longer one. In our study we show that the translation of the human MC3R protein starts at the evolutionary conserved ATG codon which results in a shorter protein than previously assumed. Conclusion: The polymorphism T6K is not located in the coding region of the human MC3R and has no influence on translation initiation which makes an impact on body weight unlikely.

Melanocortin 3 Receptor Has a 5′ Exon That Directs Translation of Apically Localized Protein From the Second In-Frame ATG

“…Taken together, these results indicate that translation of MC3R is initiated at the second ATG and the full-length protein encompasses 323 amino acids. This conclusion is consistent with cross-species conservation of the second but not the first ATG of MC3R and with studies of truncated 5Ј MC3R fused to reporter protein that demonstrated preferential initiation from the second ATG (27).…”

“…Prior studies indicate that both ATGs can function as translation initiation codons and that the sequence between the first and second ATG is not critical for the ligand binding of the receptor (25,26). Recently, it has been shown that the second ATG is preferentially used as the translation initiation site in a truncated form of MC3R (27). Lack of information regarding the composition of the full-length mRNA transcript, as well as conflicting results on translation initiation, prompted us to identify native transcription and translation start sites to gain a more complete understanding of the MC3R protein.…”

A mouse model for a partially inactive obesity-associated human MC3R variant

“…In vitro studies have suggested there is decreased MC3R protein expression and therefore partial inactivation of the MC3R after transient transfections of C17A+G241A MC3R 13 15 . However, other in vitro data suggest that C17A may actually reside in the 5′-UTR of the major MC3R transcript 17 18 . It is conceivable that C17A+G241A may change the balance of translation from the first and second in-frame ATGs and potentially interfere with MC3R function, for instance by altering its membrane localization 18 .…”

“…Other variants, including C17A (Thr6Lys) and G241A (Val81Ile) have not been found individually to affect signal transduction 12 or be associated with body weight 6 8 9 10 . We and others have reported that homozygosity for the C17A+G241A MC3R haplotype is associated with childhood obesity and higher fat mass than observed for wild type or heterozygous children 13 14 15 , although this result has not been replicated among adults 16 and some in vitro data suggest that the major start site for translation for MC3R may begin at a second in-frame ATG that is downstream of C17A, placing C17A in the 5′ untranslated region 17 18 . However, some in vitro studies have found translation is possible from the first ATG 19 and have suggested that the C17A+G241A hMC3R may be partially inactive, with significantly fewer surface receptor binding sites, decreased signal transduction and less protein expression 13 15 .…”

Polymorphisms and mutations in the melanocortin-3 receptor and their relation to human obesity

“…Park et al reported that the full-length 5’ UTR directs utilization of the second in-frame ATG as the primary translation start site instead of the canonically-assumed first ATG [13]. This leads to a protein transcript which is 37 codons shorter than the classical MC3R transcript [15]; although other research suggests that translation can occur from the first ATG site as well [16]. These studies suggested the possibility of transcript heterogeneity for both human MC3R and murine Mc3r that could potentially enable tissue-specific gene regulation.…”