SAT026 Melanocortin Receptors In The Earliest Chordates?

Abstract Disclosure: R. Ji: None. S. Jiang: None. G. Kleinau: None. P. Scheerer: None. Y. Tao: None. Melanocortin receptors (MCRs) genes have been identified to be functional in the genome of early chordates, e.g. the cyclostomata, around 360 million years ago. Whether it appears in the most ancient chordates, such as cephalochordate and urochordata (500 million years ago), however, remained unknown until now. Herein, therefore, we studied experimentally four putative, sequence based predicted melanocortin-4 receptor (mc4r) like receptors and one putative MC1R-like receptor from urochordata and cephalochordate (from NCBI database), including Styela clava (sc), Ciona intestinalis (ci), Branchiostoma floridae (fl), and Branchiostoma belcheri (bb). No genes coding the endogenous MCR ligands were observed in these species. We further expressed these putative receptor plasmids in HEK293T cells and stimulated with α-melanocyte stimulating hormone (α-MSH) and synthetic MCR ligands to verify whether these receptors are functional MCRs. Our results showed that four receptors (so far named flMC4R, bbMC4R-1, bbMC4R-2, and ciMC1R) had high cell surface expression in HEK293T cells, whereas scMC4R had low cell surface expression. Four receptors (except scMC4R) showed high basal cAMP signaling, suggesting that these receptors are likely coupled to the stimulatory Gs protein. However, no specific binding to the superpotent α-MSH analog, NDP-MSH, were observed at these five receptors, and in accordance they had no α-MSH-induced cAMP signaling coupled to Gs stimulation. Additionally, all five receptors had low basal and no α-MSH-stimulated ERK1/2 signaling. Four human MC4R inverse agonists, including Agouti-related protein and three small molecular compounds (Ipsen5i, MCL0020, and ML00253764), had no effect on cAMP and ERK1/2 signaling of the four predicted MC4R-like receptors. In summary, our results suggested that the four predicted MCR-like receptors (except scMC4R) in ancient chordates are indeed functional receptors, with high constitutive activity, but they are all not MCR-like receptors based on our experimental data with common endogenous and synthetic MCR ligands. We hypothesize that these receptors might be MCR-like receptors, in that receptors might appear earlier than appearance of endogenous ligands; or these receptors might be other ancient G protein-coupled receptors (GPCRs) with other unidentified ligands. However, further studies are needed to identify the ancient GPCR system, which may include studies on the appearance of ligands or precursors. Presentation: Saturday, June 17, 2023