Defining the Contribution of MC1R Physiological Ligands to ATR Phosphorylation at Ser435, a Predictor of DNA Repair in Melanocytes

The melanocortin 1 receptor (MC1R), a G(S)-coupled receptor that signals through cAMP and protein kinase A (PKA), regulates pigmentation, adaptive tanning, and melanoma resistance. MC1R-cAMP signaling promotes PKA-mediated phosphorylation of ataxia telangiectasia and rad3-related (ATR) at Ser435 (ATR-pS435), a modification that enhances nucleotide excision repair (NER) by facilitating recruitment of the XPA protein to sites of UV-induced DNA damage. High-throughput methods were developed to quantify ATR-pS435, measure XPA-photodamage interactions, and assess NER function. We report that melanocyte-stimulating hormone (alpha-MSH) or ACTH induce ATR-pS435, enhance XPA's association with UV-damaged DNA and optimize melanocytic NER. In contrast, MC1R antagonists agouti signaling protein (ASIP) or human beta-defensin 3 (HBD3) interfere with ATR-pS435 generation, impair the XPA-DNA interaction, and reduce DNA repair. Although ASIP and HBD3 each blocked alpha-MSH-mediated induction of the signaling pathway, only ASIP depleted basal ATR-pS435. Our findings confirm that ASIP diminishes agonist-independent MC1R basal signaling whereas HBD3 is a neutral MC1R antagonist that blocks activation by melanocortins. Furthermore, our data suggest that ATR-pS435 may be a useful biomarker for the DNA repair-deficient MC1R phenotype.