Regulación de la señalización no canónica por el receptor de melanocortinas 1 humano

Abstract

The melanocortin 1 receptor (MC1R) expressed in epidermal melanocytes is a major determinant of the cutaneous responses to ultraviolet radiation. MC1R signals primarily through the cAMP pathway and the ERK1/2 module. Its functional coupling is regulated by general mechanisms acting on most if not all GPCRs, but also by non-canonical mechanisms such as modulation by allelic variants of the MC1R gene or interactions with accessory proteins like Mahogunin (MGRN1). This protein displays E3-ubiquitin ligase activity and competes with Gs for binding with MC1R. The mahoganoid mutation abolishes mouse Mgrn1 expression and yields mice with darker furs, high embryonic lethality and propensity to cardiopathies and spongiform neurodegeneration. With this background in mind, our aims were to study a) the function of MC1R allelic variants associated with human pigmentation phenotypes and b) the role of MGRN1 in melanin pigmentation. We characterized the function of two natural MC1R mutants identified in hypopigmented individuals born in Pakistan: one premature truncation (p.Y298*) and a one-codon deletion (p.V174del). Both mutants showed reduced intracellular stability and impaired anterograde trafficking, with retention in intracellular compartments and lower expression on the plasma membrane. Moreover, p.Y298* was unable to bind agonists, whereas p.V174del showed high affinity for melanocortin peptides. Therefore, both forms are functionally impaired, with partial or complete loss-of-function for p.V174del and p.Y298*, respectively. The role of MGRN1 as a regulator of melanin pigmentation was analyzed using control mouse melanocytes (melan-a6 cells), Mgrn1-null mahoganoid melanocytes (melan-md1 cells), clones of melan-a6 melanocytes or B16F10 mouse melanoma cells where Mgrn1 expression was abolished by CRISPR/Cas9, as well as human melanoma cells where MGRN1 expression was repressed by means of siRNA. Loss of Mgrn1 expression in mouse melanocytes led to a more differentiated phenotype with increased dendricity and melanin contents. Ultrastructural analysis showed that Mgrn1-null cells had more melanosomes and that highly melanized stage IV melanosomes were more abundant than in melan-a6 melanocytes. Absence of Mgrn1 also increased tyrosinase activity measured in live cells, but not mRNA or protein levels, or the enzymatic tyrosinase activity measured in vitro using cell-free extracts. This activation of tyrosinase in situ was due to an increased melanosomal pH, as measured by acidic organelle staining with two different probes, DAMP or AO. Mgrn1 overexpression acidified the lumen of lysosome-related organelles (LRO) in HEK293T cells and decreased the activity of tyrosinase expressed ectopically in these cells. Taken together, these data suggest that Mgrn1 is a yet uncharacterized regulator of the pH of lysosomal lineage organelles that contributes to the biogenesis, maturation, transport and regulation of the luminal pH of melanosomes. Moreover, since these results were confirmed in a heterologous system, this new function of Mgrn1 as a regulator of the luminal pH of acidic organelles may not be restricted to melanocytic cells but may extend to the LROs of other cell types. Comparison of gene expression profiles of melan-a6 and melan-md1 cells confirmed the role of Mgrn1 in the regulation of melanosomal pH and pointed to Mcoln3, Atp6v0d2 y Ctns as genes overexpressed in melan-md1 cells potentially involved in this process. We verified induction of these genes in melan-a6 cells following silencing of Mgrn1 by CRISPR/Cas9 or upon treatment with Mgrn1-directed siRNA. Functional validation experiments suggested that Mcoln3 mediates the effect of Mgrn1 on the melanosomal pH, since silencing Mcoln3 expression in melan-md1 cells decreased tyrosinase activity as well as the pH of the melanosomal lumen, as estimated with DAMP.