ESPE Yearbook of Paediatric Endocrinology

J Cell Rep. 2021 Mar 23;34(12):108862. 10.1016/j.celrep.2021.108862. https://pubmed.ncbi.nlm.nih.gov/33761344/

This study demonstrates that MC4R variants found in humans affect receptor endocytosis, trafficking and dimerization and thus reveal multiple cellular mechanisms involved in weight regulation. The findings contributes to our understanding of the complex mechanisms that lie behind the melanocortin 4 receptor’s (MC4R) pivotal role in weight regulation. Stimulation of MC4R, a G-protein coupled receptor, is considered to be one of the key factors that reduces appetite and regulates energy homeostasis and body weight. It was previously shown that the binding of pro-opiomelanocortin (POMC) derived melanocyte-stimulating hormone (MSH) to the MC4R leads to increased production of cyclic AMP (cAMP) (1). We are aware of several G-protein related pathways that can be ‘drugged’ (2) and meanwhile MC4R is a prime target for anti-obesity drugs (3). Informed by the recently published 3D structure of the MC4R (4) novel therapeutic methods to target MC4R can be more efficiently investigated and developed.

These authors aimed to investigate the cellular functioning and trafficking of MC4R by looking at effects of several naturally occurring MC4R variants when experimentally expressed in HEK293 cells, which do not endogenously express MC4R. 48 rare MC4R variants from previously published large cohorts (minor allele frequency [MAF], <1%) (5-7) and 2 more common variants (MAF 1-2%) that have previously been associated with obesity protection (8-11) were studied.

To quantitatively assess and monitor the intracellular and extracellular interaction/signalization of different MC4R pathways, such as plasma membrane (PM) expression, β- arrestin recruitment, G-α interaction, MAPK (mitogen activated protein kinases)/ERK (extracellular signal-regulated kinases) phosphorylation, homodimerization and early (recycling) or late (degradation) endocytosis, enhanced bystander bioluminescence resonance energy transfer (ebBRET) sensors (12) were applied. The effects of these 50 MC4R variants were functionally categorized as either gain of function (GOF) and/or loss of function (LOF) for each of their respective cellular interaction pathways. Interestingly, some pathways might be able to regulate MC4R activity independent of cAMP production. 19 MC4R variants previously shown to be similar to WT or to have none/or limited effects on cAMP production (>85% of WT cAMP production) (13) are shown here to impair one or more pathways. Based on these results, there are obviously novel ways to target MC4R. As an example, targeting the homodimerization process with an allosteric modulator might be an option similar to several experimental therapies that are currently under investigation for central nervous system disorders (14).

By dissecting mechanisms that regulate MC4R with naturally occurring human variants, this study expands our knowledge of MC4R functionality. We believe that further human as well as transgenic animal and cell model studies are needed to further examine the relevance of these new mechanisms. A recent study (3) (see paper 11.2), has shown the interaction between setmelanotide and several naturally occurring human MC4R variants by using cryogenic electron microscopy and thereby helps to better understand the core functioning of MC4R and to discover future pharmacological treatments.

References: 1. Schwartz MW, Woods SC, Porte D Jr, Seeley RJ, Baskin DG. Central nervous system control of food intake. Nature. 2000 Apr 6;404(6778):661–71. doi: 10.1038/35007534. 2. Hauser AS, Attwood MM, Rask-Andersen M, Schiöth HB, Gloriam DE. Trends in GPCR drug discovery: new agents, targets and indications. Nat Rev Drug Discov. 2017 Dec;16(12):829–842. doi: 10.1038/nrd.2017.178. 3. Israeli H, Degtjarik O, Fierro F, Chunilal V, Gill AK, Roth NJ, Botta J, Prabahar V, Peleg Y, Chan LF, Ben-Zvi D, McCormick PJ, Niv MY, Shalev-Benami M. Structure reveals the activation mechanism of the MC4 receptor to initiate satiation signaling. Science. 2021 May 21;372(6544):808–814. doi:10.1126/science.abf7958. 4. Yu J, Gimenez LE, Hernandez CC, Wu Y, Wein AH, Han GW, McClary K, Mittal SR, Burdsall K, Stauch B, Wu L, Stevens SN, Peisley A, Williams SY, Chen V, Millhauser GL, Zhao S, Cone RD, Stevens RC. Determination of the melanocortin-4 receptor structure identifies Ca2+ as a cofactor for ligand binding. Science. 2020 Apr 24;368(6489):428–433. doi: 10.1126/science. aaz8995. 5. Farooqi IS, Keogh JM, Yeo GS, Lank EJ, Cheetham T, O’Rahilly S. Clinical spectrum of obesity and mutations in the melanocortin 4 receptor gene. N Engl J Med. 2003 Mar 20;348(12):1085–95. doi: 10.1056/NEJMoa022050. 6. Hinney A, Bettecken T, Tarnow P, Brumm H, Reichwald K, Lichtner P, Scherag A, Nguyen TT, Schlumberger P, Rief W, Vollmert C, Illig T, Wichmann HE, Schäfer H, Platzer M, Biebermann H, Meitinger T, Hebebrand J. Prevalence, spectrum, and functional characterization of melanocortin-4 receptor gene mutations in a representative population-based sample and obese adults from Germany. J Clin Endocrinol Metab. 2006 May;91(5):1761–9. doi: 10.1210/jc.2005-2056. 7. Stutzmann F, Tan K, Vatin V, Dina C, Jouret B, Tichet J, Balkau B, Potoczna N, Horber F, O’Rahilly S, Farooqi IS, Froguel P, Meyre D. Prevalence of melanocortin-4 receptor deficiency in Europeans and their age-dependent penetrance in multigenerational pedigrees. Diabetes. 2008 Sep;57(9):2511–8. doi: 10.2337/db08-0153. 8. Lotta LA, Mokrosiński J, Mendes de Oliveira E, et al. Human Gain-of-Function MC4R Variants Show Signaling Bias and Protect against Obesity. Cell. 2019;177(3):597–607.e9. doi: 10.1016/j.cell.2019.03.044. 9. Stutzmann F, Vatin V, Cauchi S, Morandi A, Jouret B, Landt O, Tounian P, Levy-Marchal C, Buzzetti R, Pinelli L, Balkau B, Horber F, Bougnères P, Froguel P, Meyre D. Non-synonymous polymorphisms in melanocortin-4 receptor protect against obesity: the two facets of a Janus obesity gene. Hum Mol Genet. 2007 Aug 1;16(15):1837–44. doi: 10.1093/hmg/ddm132.10. Wang D, Ma J, Zhang S, Hinney A, Hebebrand J, Wang Y, Wang HJ. Association of the MC4R V103I polymorphism with obesity: a Chinese case-control study and meta-analysis in 55,195 individuals. Obesity (Silver Spring). 2010 Mar;18(3):573–9. doi: 10.1038/oby.2009.268.11. Young EH, Wareham NJ, Farooqi S, et al. The V103I polymorphism of the MC4R gene and obesity: population-based studies and meta-analysis of 29 563 individuals. Int J Obes (Lond). 2007;31(9):1437–1441. doi: 10.1038/sj.ijo.0803609.12. Namkung Y, Le Gouill C, Lukashova V, Kobayashi H, Hogue M, Khoury E, Song M, Bouvier M, Laporte SA. Monitoring G protein-coupled receptor and β-arrestin trafficking in live cells using enhanced bystander BRET. Nat Commun. 2016 Jul 11; 7:12178. doi: 10.1038/ncomms12178.13. Collet TH, Dubern B, Mokrosinski J, Connors H, Keogh JM, Mendes de Oliveira E, Henning E, Poitou-Bernert C, Oppert JM, Tounian P, Marchelli F, Alili R, Le Beyec J, Pépin D, Lacorte JM, Gottesdiener A, Bounds R, Sharma S, Folster C, Henderson B, O’Rahilly S, Stoner E, Gottesdiener K, Panaro BL, Cone RD, Clément K, Farooqi IS, Van der Ploeg LHT. Evaluation of a melanocortin-4 receptor (MC4R) agonist (Setmelanotide) in MC4R deficiency. Mol Metab. 2017 Oct;6(10):1321–1329. doi: 10.1016/j.molmet.2017.06.015.14. Nickols HH, Conn PJ. Development of allosteric modulators of GPCRs for treatment of CNS disorders. Neurobiol Dis. 2014 Jan; 61:55–71. doi: 10.1016/j.nbd.2013.09.013.