Pharmacological Characterization of Loss of Function Mutations of the Human Melanocortin 1 Receptor That Are Associated with Red Hair  Aneta Ringholm, Janis Klovins, Richard Rudzish, Sion Phillips, Jonathan L. Rees, Helgi B. Schiöth  Journal of Investigative Dermatology  Volume 123, Issue 5, Pages 917-923 (November 2004) DOI: 10.1111/j.0022-202X.2004.23444.x Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Radioligand binding. Competition studies showing the affinity of α-MSH to the wild-type (▪) MC1 receptor and the Asp84Glu (•), Val92Met (▴) and Arg163Gln (⧫) MC1 receptor mutants. Competition curves are obtained using COS-1 cells transfected with the various MC1 receptors using a fixed concentration of nM [125I][Nle4, D-Phe7]α-MSH and varying concentrations of α-MSH. Lines represent the computer-modelled best fit of the data assuming that ligands bound to one-site. Journal of Investigative Dermatology 2004 123, 917-923DOI: (10.1111/j.0022-202X.2004.23444.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Second messenger study. Generation of cAMP in respons to α-MSH in HEK293-EBNA cells transfected with wild-type (▪) MC1 receptor and the different MC1 receptor mutants: Asp84Glu (•), Val92Met (▴), Arg163Gln (⧫) and Asp294His (□). Each point represents the average±SEM. Untransfected HEK293-EBNA (○) cells showed no AC-activity in response to α-MSH. The small figure, in the right upper corner, is an enlargement of the three curves with lowest cAMP response (HEK293-EBNA (○), Asp84Glu (•) and Asp294His (□)) from the main figure. The cAMP assay was performed in duplicates and repeated three times for each receptor. Journal of Investigative Dermatology 2004 123, 917-923DOI: (10.1111/j.0022-202X.2004.23444.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Two-dimensional model of human MC1 receptor (Accession number Q01726). The amino acids are shown in the circles. The residues shown in filled circles indicate positions for variants of the human MC1 receptor, where a mutation results in amino acid exchange, whereas the residues shown in double circles shows positions for synonymous mutations, where a base pair substitution does not results in change of an amino acid. (A) The mutants show strong association with fair skin and red hair. (B) The mutants show clearly impaired ability to stimulate cAMP production in response to α-MSH stimulation. (C) The mutants show weak or no association to fair skin and/or red hair. The positions for trans-membrane regions, cytoplasmatic loops (IL) and extracellular loops (EL) were drawn using crystal structure of bovine rhodopsin as a template (Palczewski et al, 2000). Alignment of the human MC1 receptor and bovine rhodopsin was done using Clustal W (1.7) software. Journal of Investigative Dermatology 2004 123, 917-923DOI: (10.1111/j.0022-202X.2004.23444.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions