Figure 2

Functional characterization of p.Ala291Asp and p.Gly248Cys variants suggests loss of function

(A) GFP-tagged wild-type (WT) human MC1R and p.Gly248Cys variant were overexpressed in A375 (human melanocytes) for 24 h, followed by confocal microscopy. The variant leads to the partial accumulation of mutant protein in the cytosol. WGA, wheat germ agglutinin. Scale bar, 10 μM.

(B) Molecular signaling activation with α-MSH (10⁻⁶ M) for 60 min in HEK293T cells overexpressing WT and p.Ala291Asp and p.Gly248Cys variants showed significant downregulation in MITF level. Quantification of MITF level (normalized with GAPDH) in the presence of α-MSH (10⁻⁶ M) for 60 min; n = 4.

(C) The pigmentation deficit, due to morpholino-based reduction of endogenous mc1r, was rescued by overexpression of human WT MC1R in zebrafish embryos. Co-injection of MO with p.Ala291Asp and p.Gly248Cys variants resulted in partial or no restoration of pigmentation levels, respectively. Scale bar, 2000 μM.

(D) Quantification of pigmentation levels, measured as a mean gray value in the head region of the injected embryos. Statistical significance was evaluated by Student’s t test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.