Abstract
Purpose
Melanocortin-3 receptor (MC3R), melanocortin-4 receptor (MC4R), and a recently identified melanocortin-2 receptor accessory protein 2 (MRAP2), are highly expressed in hypothalamus and coordinately regulate energy homeostasis, but the single cellular transcriptome of melanocortin system remains unknown. Several infrequent MRAP2 variants are reported from severe obese human patients but the mechanisms on how they affect melanocortin signaling are unclear.
Methods
First, we performed in silico analysis of mouse hypothalamus RNA sequencing datasets at single-cell resolution from two independent studies. Next, we inspected the three-dimensional conformational alteration of three mutations on MRAP2 protein. Finally, the influence of MRAP2 variants on MC3R and MC4R signaling was analyzed in vitro.
Results
(1) We confirmed the actual co-expression of Mrap2 with Mc3r and Mc4r, and demonstrated more broad distribution of Mrap2-positive neuronal populations than Mc3r or Mc4r in mouse hypothalamus. (2) Compared with wild-type MRAP2, MRAP2N88Y, and MRAP2R125C showed impaired α-MSH-induced MC4R or MC3R stimulation. (3) MRAP2N88Yexhibited enhanced interaction with MC4R protein and its own.
Conclusions
This is the first dedicated description of single-cell transcriptome signature of Mrap2, Mc3r, and Mc4r in the central nerve system and the first evidence describing the unique dimer formation, conformational change, and pharmacological effect of MRAP2 mutations on MC3R signaling.
Similar content being viewed by others
References
P.M. Hinkle, J.A. Sebag, Structure and function of the melanocortin2 receptor accessory protein (MRAP). Mol. Cell. Endocrinol. 300, 25–31 (2009)
T.R. Webb, A.J. Clark, Minireview: the melanocortin 2 receptor accessory proteins. Mol. Endocrinol. (Baltim., Md.) 24, 475–484 (2010)
D.S. Jackson, S. Ramachandrappa, A.J. Clark, L.F. Chan, Melanocortin receptor accessory proteins in adrenal disease and obesity. Front. Neurosci. 9, 213 (2015)
A.S. Chen, D.J. Marsh, M.E. Trumbauer, E.G. Frazier, X.M. Guan, H. Yu, C.I. Rosenblum, A. Vongs, Y. Feng, L. Cao, J.M. Metzger, A.M. Strack, R.E. Camacho, T.N. Mellin, C.N. Nunes, W. Min, J. Fisher, S. Gopal-Truter, D.E. MacIntyre, H.Y. Chen, L.H. Van der Ploeg, Inactivation of the mouse melanocortin-3 receptor results in increased fat mass and reduced lean body mass. Nat. Genet. 26, 97–102 (2000)
D. Huszar, C.A. Lynch, V. Fairchild-Huntress, J.H. Dunmore, Q. Fang, L.R. Berkemeier, W. Gu, R.A. Kesterson, B.A. Boston, R.D. Cone, F.J. Smith, L.A. Campfield, P. Burn, F. Lee, Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell 88, 131–141 (1997)
M. Mencarelli, G.E. Walker, S. Maestrini, L. Alberti, B. Verti, A. Brunani, M.L. Petroni, M. Tagliaferri, A. Liuzzi, A.M. Di Blasio, Sporadic mutations in melanocortin receptor 3 in morbid obese individuals. Eur. J. Human. Genet. : EJHG 16, 581–586 (2008)
Y.S. Lee, L.K. Poh, B.L. Kek, K.Y. Loke, The role of melanocortin 3 receptor gene in childhood obesity. Diabetes 56, 2622–2630 (2007)
I.S. Farooqi, J.M. Keogh, G.S. Yeo, E.J. Lank, T. Cheetham, S. O’Rahilly, Clinical spectrum of obesity and mutations in the melanocortin 4 receptor gene. New Engl. J. Med. 348, 1085–1095 (2003)
C. Lubrano-Berthelier, C. Le Stunff, P. Bougneres, C. Vaisse, A homozygous null mutation delineates the role of the melanocortin-4 receptor in humans. J. Clin. Endocrinol. Metab. 89, 2028–2032 (2004)
Y. Bromberg, J. Overton, C. Vaisse, R.L. Leibel, B. Rost, In silico mutagenesis: a case study of the melanocortin 4 receptor. FASEB J. : Off. Publ. Fed. Am. Soc. Exp. Biol. 23, 3059–3069 (2009)
P. You, H. Hu, Y. Chen, Y. Zhao, Y. Yang, T. Wang, R. Xing, Y. Shao, W. Zhang, D. Li, H. Chen, M. Liu, Effects of melanocortin 3 and 4 receptor deficiency on energy homeostasis in rats. Sci. Rep. 6, 34938 (2016)
T.P. Braun, B. Orwoll, X. Zhu, P.R. Levasseur, M. Szumowski, M.L. Nguyen, M.L. Bouxsein, R.F. Klein, D.L. Marks, Regulation of lean mass, bone mass, and exercise tolerance by the central melanocortin system. PLoS ONE 7, e42183 (2012)
S. Roy, S.J. Roy, S. Pinard, M.J. Agulleiro, J.M. Cerda-Reverter, J.L. Parent, N. Gallo-Payet, The C-terminal domains of melanocortin-2 receptor (MC2R) accessory proteins (MRAP1) influence their localization and ACTH-induced cAMP production. General Comp. Endocrinol. 176, 265–274 (2012)
J.A. Sebag, P.M. Hinkle, Melanocortin-2 receptor accessory protein MRAP forms antiparallel homodimers. Proc. Natl Acad. Sci. USA 104, 20244–20249 (2007)
T.R. Webb, L. Chan, S.N. Cooray, M.E. Cheetham, J.P. Chapple, A.J. Clark, Distinct melanocortin 2 receptor accessory protein domains are required for melanocortin 2 receptor interaction and promotion of receptor trafficking. Endocrinology 150, 720–726 (2009)
L.F. Chan, T.R. Webb, T.T. Chung, E. Meimaridou, S.N. Cooray, L. Guasti, J.P. Chapple, M. Egertova, M.R. Elphick, M.E. Cheetham, L.A. Metherell, A.J. Clark, MRAP and MRAP2 are bidirectional regulators of the melanocortin receptor family. Proc. Natl Acad. Sci. USA 106, 6146–6151 (2009)
M. Asai, S. Ramachandrappa, M. Joachim, Y. Shen, R. Zhang, N. Nuthalapati, V. Ramanathan, D.E. Strochlic, P. Ferket, K. Linhart, C. Ho, T.V. Novoselova, S. Garg, M. Ridderstrale, C. Marcus, J.N. Hirschhorn, J.M. Keogh, S. O’Rahilly, L.F. Chan, A.J. Clark, I.S. Farooqi, J.A. Majzoub, Loss of function of the melanocortin 2 receptor accessory protein 2 is associated with mammalian obesity. Sci. (New Y., N. Y.) 341, 275–278 (2013)
J.A. Sebag, C. Zhang, P.M. Hinkle, A.M. Bradshaw, R.D. Cone, Developmental control of the melanocortin-4 receptor by MRAP2 proteins in zebrafish. Sci. (New Y., N. Y.) 341, 278–281 (2013)
J. Zhang, X. Li, Y. Zhou, L. Cui, J. Li, C. Wu, Y. Wan, J. Li, Y. Wang, The interaction of MC3R and MC4R with MRAP2, ACTH, alpha-MSH and AgRP in chickens. J. Endocrinol. 234, 155–174 (2017)
J.M. Cerda-Reverter, M.J. Agulleiro, R. Cortes, E. Sanchez, R. Guillot, E. Leal, B. Fernandez-Duran, S. Puchol, M. Eley, Involvement of melanocortin receptor accessory proteins (MRAPs) in the function of melanocortin receptors. General Comp. Endocrinol. 188, 133–136 (2013)
L. Schonnop, G. Kleinau, N. Herrfurth, A.L. Volckmar, C. Cetindag, A. Muller, T. Peters, S. Herpertz, J. Antel, J. Hebebrand, H. Biebermann, A. Hinney, Decreased melanocortin-4 receptor function conferred by an infrequent variant at the human melanocortin receptor accessory protein 2 gene. Obesity (Silver Spring, Md.) 24, 1976–1982 (2016)
Chaly, A.L., Srisai, D., Gardner, E.E., Sebag, J.A., The melanocortin receptor accessory protein 2 promotes food intake through inhibition of the prokineticin receptor-1, Elife 5, (2016) .
D. Srisai, T.C. Yin, A.A. Lee, A.A.J. Rouault, N.A. Pearson, J.L. Grobe, J.A. Sebag, MRAP2 regulates ghrelin receptor signaling and hunger sensing. Nat. Commun. 8, 713 (2017)
A.A.J. Rouault, A.A. Lee, J.A. Sebag, Regions of MRAP2 required for the inhibition of orexin and prokineticin receptor signaling. Biochim. Et. Biophys. Acta 1864, 2322–2329 (2017)
A.A.J. Rouault, D.K. Srinivasan, T.C. Yin, A.A. Lee, J.A. Sebag, Melanocortin receptor accessory proteins (MRAPs): Functions in the melanocortin system and beyond. BBA-Mol. Basis Dis. 1863, 2462–2467 (2017)
B.W. Jones, G.J. Song, E.K. Greuber, P.M. Hinkle, Phosphorylation of the endogenous thyrotropin-releasing hormone receptor in pituitary GH3 cells and pituitary tissue revealed by phosphosite-specific antibodies. J. Biol. Chem. 282, 12893–12906 (2007)
J. Peng, J. Xu, RaptorX: exploiting structure information for protein alignment by statistical inference. Proteins 79(Suppl 10), 161–171 (2011)
We’re living in a 3D world. Nat. Struct. Mol. Biol. 13, 93 (2006).
R.A. Romanov, A. Zeisel, J. Bakker, F. Girach, A. Hellysaz, R. Tomer, A. Alpar, J. Mulder, F. Clotman, E. Keimpema, B. Hsueh, A.K. Crow, H. Martens, C. Schwindling, D. Calvigioni, J.S. Bains, Z. Mate, G. Szabo, Y. Yanagawa, M.D. Zhang, A. Rendeiro, M. Farlik, M. Uhlen, P. Wulff, C. Bock, C. Broberger, K. Deisseroth, T. Hokfelt, S. Linnarsson, T.L. Horvath, T. Harkany, Molecular interrogation of hypothalamic organization reveals distinct dopamine neuronal subtypes. Nat. Neurosci. 20, 176–188 (2017)
R. Chen, X. Wu, L. Jiang, Y. Zhang, Single-Cell RNA-Seq Reveals Hypothalamic Cell Diversity. Cell Rep. 18, 3227–3241 (2017)
T.V. Novoselova, R. Larder, D. Rimmington, C. Lelliott, E.H. Wynn, R.J. Gorrigan, P.H. Tate, L. Guasti, S. O’Rahilly, A.J.L. Clark, D.W. Logan, A.P. Coll, L.F. Chan, S.M.G. Project, Loss of Mrap2 is associated with Sim1 deficiency and increased circulating cholesterol. J. Endocrinol. 230, 13–26 (2016)
K. Begriche, P.R. Levasseur, J.Y. Zhang, J. Rossi, D. Skorupa, L.A. Solt, B. Young, T.P. Burris, D.L. Marks, R.L. Mynatt, A.A. Butler, Genetic dissection of the functions of the melanocortin-3 receptor, a seven-transmembrane G-protein-coupled receptor, suggests roles for central and peripheral receptors in energy homeostasis. J. Biol. Chem. 286, 40771–40781 (2011)
G.M. Sutton, D. Perez-Tilve, R. Nogueiras, J. Fang, J.K. Kim, R.D. Cone, J.M. Gimble, M.H. Tschop, A.A. Butler, The melanocortin-3 receptor is required for entrainment to meal intake. J. Neurosci. 28, 12946–12955 (2008)
G.M. Sutton, J.L. Trevaskis, M.W. Hulver, R.P. McMillan, N.J. Markward, M.J. Babin, E.A. Meyer, A.A. Butler, Diet-genotype interactions in the development of the obese, insulin-resistant phenotype of C57BL/6J mice lacking melanocortin-3 or-4 receptors. Endocrinology 147, 2183–2196 (2006)
K.L.J. Ellacott, J.G. Murphy, D.L. Marks, R.D. Cone, Obesity-induced inflammation in white adipose tissue is attenuated by loss of melanocortin-3 receptor signaling. Endocrinology 148, 6186–6194 (2007)
J.A. Sebag, P.M. Hinkle, Regulation of G protein-coupled receptor signaling: specific dominant-negative effects of melanocortin 2 receptor accessory protein 2. Sci. Signal. 3, ra28 (2010)
Acknowledgements
We would like to thank Xin Xie for providing the pCRE-luc plasmids. We thank Wenbiao Chen for helpful discussion and valuable comments on the manuscript preparation. The work was supported by grants from National Key Research and Development Program of China (Grant No. 2017YFA0103900, 2017YFA0103902, and 2016YFA0102200); The National Natural Science Foundation of China (Grant No. 81570760 and 31771283); One Thousand Youth Talents Program of China to Chao Zhang and Bing Luan; The Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning (No. A11323); The Shanghai Rising-Star Program (Grant No 15QA1403600); the Fundamental Research Funds for the Central Universities of Tongji University.
Author contributions
J.L., L.L., L.G., and C.Z. conceived and designed the experiments; J.L., L.L., X.J., B.X., and L.P. performed experiments; J.L., L.L., X.J., B.X., S.L., W.Z., and C.Z. analyzed data; J.L., L.L., C.Z., B.L., L.G., and C.Z. interpreted results of experiments; J.L., L.L., and C.Z. approved final version of manuscript.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Electronic supplementary material
Rights and permissions
About this article
Cite this article
Liang, J., Li, L., Jin, X. et al. Pharmacological effect of human melanocortin-2 receptor accessory protein 2 variants on hypothalamic melanocortin receptors. Endocrine 61, 94–104 (2018). https://doi.org/10.1007/s12020-018-1596-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12020-018-1596-2