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 GS-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 (α-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 β-defensin 3 (HBD3) interfere with ATR-pS435 generation， impair the XPA-DNA interaction， and reduce DNA repair. Although ASIP and HBD3 each blocked α-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.