title: The E92K melanocortin 1 receptor mutant induces cAMP production and arrestin recruitment but not ERK activity indicating biased constitutive signaling.
abstract: BACKGROUND
  The melanocortin 1 receptor (MC1R) constitutes a key regulator of melanism. Consequently, many naturally-occurring MC1R mutations are associated with a change in color. An example is the Glu-to-Lys substitution found at position II:20/2.60 in the top of transmembrane helix II which has been identified in melanic mice and several other species. This mutation induces a pronounced increase in MC1R constitutive activity suggesting a link between constitutive activity and melanism which is corroborated by the attenuation of α-melanocyte stimulating hormone (αMSH) induced activation. However, the mechanism by which the mutation induces constitutive activity is currently not known.
  
  
  RESULTS
  Here we characterize the constitutive activity, cell surface expression and internalization of the mouse mutant, Mc1r E92K. As previously reported, only positively charged residues at position II:20/2.60 induced an increase in constitutive activity as measured by cAMP accumulation and CREB activation. Furthermore, the mutation induced a constitutive recruitment of β-arrestin. This phenomenon is only observed in MC1R, however, as the equivalent mutations in MC2-5R had no effect on receptor signaling. Interestingly, the mutation did not induce constitutive ERK1/2 phosphorylation or increase the internalization rate indicating the constitutive activity to be biased. Finally, to identify regions of importance for the increased constitutive activity of Mc1r E92K, we employed a chimeric approach and identified G102 and L110 in the extracellular loop 1 to be selectively important for the constitutive activity as this, but not αMSH-mediated activation, was abolished upon Ala substitution.
  
  
  CONCLUSIONS
  It is concluded that the E92K mutation induces an active conformation distinct from that induced by αMSH and that the extracellular loop 1 is involved in maintaining this conformational state. In turn, the results suggest that in MC1R, which lacks an extracellular loop 2, the first extracellular loop may play a more prominent role during receptor activation than in general.
authors.affiliation: Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Tau
authors.initials: T
authors.lastname: Benned-Jensen
authors.forename: Jacek
authors.initials: J
authors.lastname: Mokrosinski
authors.forename: Mette M
authors.initials: MM
authors.lastname: Rosenkilde
mesh_terms: D000818:Animals; D019393:Arrestin / Q000378:metabolism*; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D044102:Receptor, Melanocortin, Type 1 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D000521:alpha-MSH / Q000235:genetics / Q000378:metabolism
chemical_list: D019393:Arrestin; D044102:Receptor, Melanocortin, Type 1; D000521:alpha-MSH; D000242:Cyclic AMP; D048049:Extracellular Signal-Regulated MAP Kinases