Engineering the melanocortin-4 receptor to control constitutive and ligand-mediated G(S) signaling in vivo

PLoS One. 2007 Aug 1;2(7):e668. doi: 10.1371/journal.pone.0000668.

Abstract

The molecular and functional diversity of G protein-coupled receptors is essential to many physiological processes. However, this diversity presents a significant challenge to understanding the G protein-mediated signaling events that underlie a specific physiological response. To increase our understanding of these processes, we sought to gain control of the timing and specificity of G(s) signaling in vivo. We used naturally occurring human mutations to develop two G(s)-coupled engineered receptors that respond solely to a synthetic ligand (RASSLs). Our G(s)-coupled RASSLs are based on the melanocortin-4 receptor, a centrally expressed receptor that plays an important role in the regulation of body weight. These RASSLs are not activated by the endogenous hormone alpha-melanocyte-stimulating hormone but respond potently to a selective synthetic ligand, tetrahydroisoquinoline. The RASSL variants reported here differ in their intrinsic basal activities, allowing the separation of the effects of basal signaling from ligand-mediated activation of the G(s) pathway in vivo. These RASSLs can be used to activate G(s) signaling in any tissue, but would be particularly useful for analyzing downstream events that mediate body weight regulation in mice. Our study also demonstrates the use of human genetic variation for protein engineering.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cell Line
  • Cell Membrane / drug effects
  • Cell Membrane / metabolism
  • Cyclic AMP / metabolism
  • Heterozygote
  • Humans
  • Kidney
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Obesity / genetics*
  • Point Mutation
  • Protein Conformation
  • Protein Engineering / methods
  • Receptor, Melanocortin, Type 4 / chemistry
  • Receptor, Melanocortin, Type 4 / genetics*
  • Receptor, Melanocortin, Type 4 / metabolism
  • Tetrahydroisoquinolines / pharmacology
  • Transfection / methods
  • Triazoles / pharmacology
  • alpha-MSH / pharmacology

Substances

  • MC4R protein, human
  • N-(1,2,3,4-tetrahydroisoquinolinium-3-ylcarbonyl)-1-(4-chlorobenzyl)-2-(4-cyclohexyl-4-(1H-1,2,4-triazol-1-ylmethyl)piperidin-1-yl)-2-oxoethylamine
  • Receptor, Melanocortin, Type 4
  • Tetrahydroisoquinolines
  • Triazoles
  • alpha-MSH
  • Cyclic AMP