The E92K Melanocortin 1 Receptor Mutant Induces cAMP Production and Arrestin Recruitment but Not ERK Activity Indicating Biased Constitutive Signaling

Abstract

BackgroundThe 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.Methodology/Principal FindingsHere 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/SignificanceIt 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.

Defect in MAPK Signaling As a Cause for Monogenic Obesity Caused By Inactivating Mutations in the Melanocortin-4 Receptor Gene

“…Because the basal activities in the Gs-cAMP pathway for the three mutations, R165G, R165W and C172R, are not increased compared to the WT MC4R 43 , 56 , but they were constitutively active in the ERK1/2 signaling pathway, therefore they had biased constitutive activity. Previously, in the melanocortin-1 receptor, a mutant receptor constitutively active in the Gs-cAMP pathway is not constitutively active in the ERK1/2 pathway, also representing a case of biased constitutive signaling 57 .…”

Extracellular Disulfide Bridges Serve Different Purposes in Two Homologous Chemokine Receptors, CCR1 and CCR5

“…Intriguingly, ECL-2 is the largest (although absent in certain class A receptors such as the metal-ion chelator receptors (Benned-Jensen et al, 2011) and the most divergent of all three extracellular loops, yet among chemokine receptors we here show that the length of ECL-2 is quite conserved (Fig. 1C).…”

Identification and pharmacological analyses of eight naturally occurring caprine melanocortin-1 receptor mutations in three different goat breeds

“…Impaired ligand-stimulated ERK1/2 activation was identified for K226E, exhibiting biased signaling. In the mouse MC1R, a mutant receptor constitutively active in the cAMP pathway is not active in both basal and a-MSH induced ERK1/2 signaling, also representing a case of biased signaling (Benned-Jensen et al, 2011). Of note, biased signaling has been reported for MC4R and MC3R mutants (Breit et al, 2011;He and Tao, 2014;Mo and Tao, 2013;Mo et al, 2012;Yang et al, 2015), including biased constitutive signaling (Tao, 2014).…”