MGRN1 as a Phenotypic Determinant of Human Melanoma Cells and a Potential Biomarker

Mahogunin Ring Finger 1 (MGRN1), a ubiquitin ligase expressed in melanocytes, interacts with the α melanocyte-stimulating hormone receptor, a well-known melanoma susceptibility gene. Previous studies showed that MGRN1 modulates the phenotype of mouse melanocytes and melanoma cells, with effects on pigmentation, shape, and motility. Moreover, MGRN1 knockdown augmented the burden of DNA breaks in mouse cells, indicating that loss of MGRN1 promoted genomic instability. However, data concerning the roles of MGRN1 in human melanoma cells remain scarce. We analyzed MGRN1 knockdown in human melanoma cells. Transient MGRN1 depletion with siRNA or permanent knockdown in human melanoma cells by CRISPR/Cas9 caused an apparently MITF-independent switch to a more dendritic phenotype. Lack of MGRN1 also increased the fraction of human cells in the S phase of the cell cycle and the burden of DNA breaks but did not significantly impair proliferation. Moreover, in silico analysis of publicly available melanoma datasets and estimation of MGRN1 in a cohort of clinical specimens provided preliminary evidence that MGRN1 expression is higher in human melanomas than in normal skin or nevi and pointed to an inverse correlation of MGRN1 expression in human melanoma with patient survival, thus suggesting potential use of MGRN1 as a melanoma biomarker.This research was funded by grant SAF2018_RTI2018-094929-B-I00 financed by FEDER/Ministerio de Ciencia e Innovación—Agencia Estatal de Investigación (Spain) (to C.J.-C. and J.C.G.-B.), and by grant UPV/EHU GIU20/035 (to S.A and M.D.B.)

Molecular basis of the extreme dilution mottled mouse mutation: A combination of coding and non-coding genomic alterations

8 pages, 4 figures.-- PMID: 15572362 [PubMed].-- The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EBI Data Bank with accession number(s) AY526904.-- Open Access version available at the publisher's site.Tyrosinase is the rate-limiting enzyme in melanin biosynthesis. It is an N-glycosylated, copper-containing transmembrane protein, whose post-translational processing involves intracytoplasmic movement from the endoplasmic reticulum to the Golgi and, eventually, to the melanosome. The expression of the tyrosinase (Tyr) gene is controlled by several regulatory regions including a locus control region (LCR) located 15 kb upstream from the promoter region. The extreme dilution mottled mutant mice (Tyr^c-em) arose spontaneously at the MRC Institute in Harwell (United Kingdom) from a chinchilla-mottled mutant (Tyr^c-m) stock, whose molecular basis corresponds to a rearrangement of 5'-upstream regulatory sequences including the LCR of the Tyr gene. Tyr^c-em mice display a variegated pigmentation pattern in coat and eyes, in agreement with the LCR translocation, but also show a generalized hypopigmented phenotype, not seen in Tyr^c-m mice. Genomic analyses of Tyr^c-em mice showed a C1220T nucleotide substitution within the Tyr encoding region, resulting in a T373I amino acid change, which abolishes an N-glycosylation sequon located in the second metal ion binding site of the enzyme. Tyrosinase from Tyr^c-em displayed a reduced enzymatic activity in vivo and in vitro, compared with wild-type enzyme. Deglycosylation studies showed that the mutant protein has an abnormal glycosylation pattern and is partially retained in the endoplasmic reticulum. We conclude that the phenotype of the extreme dilution mottled mouse mutant is caused by a combination of coding and noncoding genomic alterations resulting in several abnormalities that include suboptimal gene expression, abnormal protein processing, and reduced enzymatic activity.This work was supported by Spanish Ministry of Science and Technology Grants Bio2000-1653 and Bio2003-08196, Comunidad Autónoma de Madrid 08.5/0046/2003 (to L. M.), and SAF2003-03411 from the Comisión Interministerial de Ciencia y Tecnologia and Fondo Europeo de Desarrollo Regional (to J. C. G.-B.).Peer reviewe

Molecular basis of the extreme dilution mottled mouse mutation: A combination of coding and non-coding genomic alterations

8 pages, 4 figures.-- PMID: 15572362 [PubMed].-- The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EBI Data Bank with accession number(s) AY526904.-- Open Access version available at the publisher's site.Tyrosinase is the rate-limiting enzyme in melanin biosynthesis. It is an N-glycosylated, copper-containing transmembrane protein, whose post-translational processing involves intracytoplasmic movement from the endoplasmic reticulum to the Golgi and, eventually, to the melanosome. The expression of the tyrosinase (Tyr) gene is controlled by several regulatory regions including a locus control region (LCR) located 15 kb upstream from the promoter region. The extreme dilution mottled mutant mice (Tyr^c-em) arose spontaneously at the MRC Institute in Harwell (United Kingdom) from a chinchilla-mottled mutant (Tyr^c-m) stock, whose molecular basis corresponds to a rearrangement of 5'-upstream regulatory sequences including the LCR of the Tyr gene. Tyr^c-em mice display a variegated pigmentation pattern in coat and eyes, in agreement with the LCR translocation, but also show a generalized hypopigmented phenotype, not seen in Tyr^c-m mice. Genomic analyses of Tyr^c-em mice showed a C1220T nucleotide substitution within the Tyr encoding region, resulting in a T373I amino acid change, which abolishes an N-glycosylation sequon located in the second metal ion binding site of the enzyme. Tyrosinase from Tyr^c-em displayed a reduced enzymatic activity in vivo and in vitro, compared with wild-type enzyme. Deglycosylation studies showed that the mutant protein has an abnormal glycosylation pattern and is partially retained in the endoplasmic reticulum. We conclude that the phenotype of the extreme dilution mottled mouse mutant is caused by a combination of coding and noncoding genomic alterations resulting in several abnormalities that include suboptimal gene expression, abnormal protein processing, and reduced enzymatic activity.This work was supported by Spanish Ministry of Science and Technology Grants Bio2000-1653 and Bio2003-08196, Comunidad Autónoma de Madrid 08.5/0046/2003 (to L. M.), and SAF2003-03411 from the Comisión Interministerial de Ciencia y Tecnologia and Fondo Europeo de Desarrollo Regional (to J. C. G.-B.).Peer reviewe

Functional Characterization of MC1R-TUBB3 Intergenic Splice Variants of the Human Melanocortin 1 Receptor.

The melanocortin 1 receptor gene (MC1R) expressed in melanocytes is a major determinant of skin pigmentation. It encodes a Gs protein-coupled receptor activated by α-melanocyte stimulating hormone (αMSH). Human MC1R has an inefficient poly(A) site allowing intergenic splicing with its downstream neighbour Tubulin-β-III (TUBB3). Intergenic splicing produces two MC1R isoforms, designated Iso1 and Iso2, bearing the complete seven transmembrane helices from MC1R fused to TUBB3-derived C-terminal extensions, in-frame for Iso1 and out-of-frame for Iso2. It has been reported that exposure to ultraviolet radiation (UVR) might promote an isoform switch from canonical MC1R (MC1R-001) to the MC1R-TUBB3 chimeras, which might lead to novel phenotypes required for tanning. We expressed the Flag epitope-tagged intergenic isoforms in heterologous HEK293T cells and human melanoma cells, for functional characterization. Iso1 was expressed with the expected size. Iso2 yielded a doublet of Mr significantly lower than predicted, and impaired intracellular stability. Although Iso1- and Iso2 bound radiolabelled agonist with the same affinity as MC1R-001, their plasma membrane expression was strongly reduced. Decreased surface expression mostly resulted from aberrant forward trafficking, rather than high rates of endocytosis. Functional coupling of both isoforms to cAMP was lower than wild-type, but ERK activation upon binding of αMSH was unimpaired, suggesting imbalanced signaling from the splice variants. Heterodimerization of differentially labelled MC1R-001 with the splicing isoforms analyzed by co-immunoprecipitation was efficient and caused decreased surface expression of binding sites. Thus, UVR-induced MC1R isoforms might contribute to fine-tune the tanning response by modulating MC1R-001 availability and functional parameters