Fig 2 - available via license: Creative Commons Attribution 4.0 International
Content may be subject to copyright.
Electrophoretic analysis and intracellular stability of MC1R-TUBB3 isoforms. (A) Expression of canonical and chimeric MC1R proteins in heterologous HEK293T cells. HEK293T cells were transiently transfected to express Flag-labelled WT MC1R-001, Iso1 and Iso2. Cells were detergent-solubilized, electrophoresed and blotted. For MC1R detection, cell lysates were probed with an anti-Flag monoclonal antibody (upper blot). Membranes were also probed for TUBB3 (middle blot) and ERK2 (lower blot), as loading control (n = 5, representative blots are shown). (B) Electrophoretic pattern of MC1R-TUBB3 transcripts expressed in HBL human melanoma cells. Representative immunoblots for MC1R, TUBB3 and ERK2 are shown as in panel A (n = 5, representative blots are shown). (C) Intracellular stability of MC1R-TUBB3 chimeric fusion proteins in HEK293T cells. Flag-labelled MC1R-001, Iso1 and Iso2 were expressed in HEK293T cells. Cells were incubated with the protein synthesis inhibitor cycloheximide (Chx, 0.1 mM) for the times indicated, lysed and the levels of residual proteins in cell extracts were detected by Western blot. Representative immunoblots probed for MC1R-001, Iso1 or Iso2 with anti-Flag are shown. (D) Semi-log graph for calculation of half-lives. The intensity of receptor bands in the blots as in panel C was quantitated with ImageJ and the semi-log of residual signals was plotted against time. Half-life (t½) values correspond to the slope of the resulting lines.
Source publication
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...
Citations
... In fact, the 16q24.3 region has a high gene density, which adds to an inefficient polyadenylation tail in the MC1R gene, allowing the intergenic splicing of these loci with adjacent genes, for example, TUBB3 [49,50]. This intergenic splicing results in an MC1R-TUBB3 chimera, which is transcribed in a non-canonical mRNA that is translated in two functional isoforms of canonical MC1R -Iso1 and Iso2 [49,50]. ...
... region has a high gene density, which adds to an inefficient polyadenylation tail in the MC1R gene, allowing the intergenic splicing of these loci with adjacent genes, for example, TUBB3 [49,50]. This intergenic splicing results in an MC1R-TUBB3 chimera, which is transcribed in a non-canonical mRNA that is translated in two functional isoforms of canonical MC1R -Iso1 and Iso2 [49,50]. These isoforms preserve the general structure of an GPCR, being able to perform MC1R functions, and their expression appears to be induced by the action of □α-MSH, the MC1R agonist [37,50]. ...
... This intergenic splicing results in an MC1R-TUBB3 chimera, which is transcribed in a non-canonical mRNA that is translated in two functional isoforms of canonical MC1R -Iso1 and Iso2 [49,50]. These isoforms preserve the general structure of an GPCR, being able to perform MC1R functions, and their expression appears to be induced by the action of □α-MSH, the MC1R agonist [37,50]. Despite having a poor ability to activate cAMP pathway like the canonical MC1R due to its decreased expression on the cell surface, it was proposed that Iso1 and Iso2 may be involved in fine-tune of pigmentation response by playing a role in the rearrangement of the cytoskeleton that is required to initiate, propagate, and guide the dendrites of activated melanocytes [49,50]. ...
Forensic DNA phenotyping (FDP) consists of the use of methodologies for predicting externally visible characteristics (EVCs) from the genetic material of biological samples found in crime scenes and has proven to be a promising tool in aiding human identification in police activities. Currently, methods based on multiplex assays and statistical models of prediction of EVCs related to hair, skin, and iris pigmentation using panels of SNP and INDEL biomarkers have already been developed and validated by the forensic scientific community. As well as traces of pigmentation, an individual’s perceived age (PA) can also be considered an EVC and its estimation in unknown individuals can be useful for the progress of investigations. Liu and colleagues (2016) were pioneers in evidencing that, in addition to lifestyle and environmental factors, the presence of SNP and INDEL variants in the MC1R gene — which encodes a transmembrane receptor responsible for regulating melanin production — seems to contribute to an individual’s PA. The group highlighted the association between these MC1R gene polymorphisms and the PA in the European population, where carriers of risk haplotypes appeared to be up to 2 years older in comparison to their chronological age (CA).
Understanding that genotype–phenotype relationships cannot be extrapolated between different population groups, this study aimed to test this hypothesis and verify the applicability of this variant panel in the Rio Grande do Sul admixed population.
Based on genomic data from a sample of 261 volunteers representative of gaucho population and using a multiple linear regression (MLR) model, our group was able to verify a significant association among nine intronic variants in loci adjacent to MC1R (e.g., AFG3L1P, TUBB3, FANCA) and facial age appearance, whose PA was defined after age heteroclassification of standard frontal face images through 11 assessors.
Different from that observed in European populations, our results show that the presence of effect alleles (R) of the selected variants in our sample influenced both younger and older face phenotypes. The influence of each variant on PA is expressed as β values.
There are important molecular mechanisms behind the effects of MC1R locus on PA, and the genomic background of each population seems to be crucial to determine this influence.
... In this study, we found the MC1R proteins were integrally expressed in Cos7 cells based on Western blot by using anti-HA and anti-Flag tags (Supplementary fig. S4), like that in human [64]. Meanwhile, the total receptor protein expression did not differ between NQP and SQP groups based on both Western blot (Supplementary fig. ...
Reptiles can evolve adaptive colors in different environments, but relatively little is known about the genetic mechanisms. Here, we identified the MC1R gene and its association with intraspecific color variation in the lizard Phrynocephalus erythrurus. Analysis of the MC1R sequence in 143 individuals from dark South Qiangtang Plateau (SQP) and light North Qiangtang plateau (NQP) populations, revealed two amino acid sites that showed significant
differences in frequency between two areas. One SNP, corresponding to Glu183Lys residue, was found to be a highly significant outlier and differentially fixed for SQP and NQP populations. This residue is located in an extracellular area in the second small extracellular loop within the secondary structure of MC1R, which represents an “attachment pocket” part of the 3D structure. Cytological expression of MC1R alleles with the Glu183Lys replacement showed a 39 % increase in intracellular agonist-induced cyclic AMP levels and a 23.18 % greater cell surface expression of MC1R protein in the SQP relative to the NQP allele. Further in silico 3D modeling and in vitro binding experiments indicated a higher MC1R-α-MSH binding for the SQP allele, and elevated melanin synthesis. We provide an overview of how a single amino acid replacement leads to fundamental hanges in MC1R function, and hence shapes variation in dorsal pigmentation in lizards from different environments.
... Moreover, we investigated the mRNA expression levels of two other candidate genes, TCF25 and TUBB3, and no significant difference was found ( Figure S3). Research in humans has identified an MC1R-TUBB3 intergenic splice variant due to an inefficient poly-A site of MC1R (Herraiz et al., 2015). To explore whether this splice variant exists in ducks, we randomly selected the RNAseq samples of one black duck and one BM duck for read visualization ( Figure S4). ...
... The remaining genes in the candidate region, TUBB3 and TCF25, could also be responsible for plumage colour formation. Human MC1R has an inefficient poly-A site allowing for intergenic splicing with TUBB3, forming an MC1R-TUBB3 intergenic splice variant (Herraiz et al., 2015). ...
Birds are among the most colourful terrestrial vertebrates, with various plumage colours and patterns. We conducted a genome‐wide association study (GWAS) on an intercross F2 population of Pekin ducks and mallards (n = 722) and identified a 1.57‐Mb genetic region (Chr11: 20,176,480–21,750,101 bp) related to duck melanism. Fine mapping by linkage disequilibrium (LD) and FST analysis narrowed the final candidate region to a region of 22,500 bp (Chr11: 20,677,500–20,700,000 bp) including three coding genes, TCF25, MC1R and TUBB3. Combined with transcriptome and qRT‐PCR analysis, MC1R was identified as the unique genetic locus responsible for black plumage in ducks, and it was significantly more highly expressed in the feather bulbs of black ducks. We also identified 52G > A (Chr11: 20,696,354G > A) and 376G > A (Chr11: 20,696,678G > A) mutations in the MC1R coding region that have been widely studied in ducks. In addition, structural variations (SVs) were screened by nanopore sequencing, and no significant SV was found to be associated with the duck black plumage trait. However, we identified four novel single nucleotide polymorphisms in the MC1R regulator region (Chr11: 20,678,412G > A, Chr11: 20,679,236G > A, Chr11: 20,692,496 A > G and Chr11: 20,692,791 A > G) that had a strong association with the black plumage phenotype of ducks and combined with potential changes in transcription binding affinities. The luciferase reporter gene assay demonstrated that Chr11: 20,678,412G > A and Chr11: 20,679,236G > A led to significant promoter activity changes. Our research emphasizes the importance of MC1R regulatory region mutation in determining the duck black plumage phenotype, and these results expand our understanding of the genetic mechanism underlying duck plumage colour.
... Positions based on human genome GCRh38/hg38 assembly. signaling (Table 1; Figure 2) (Dalziel et al., 2011;Herraiz et al., 2015) (reviewed in Herraiz et al., 2017). Further investigation into the TUBB3 locus using alternative sequencing techniques may need to be performed to further validate these TUBB3 transcripts, and to better understand the prevalence of this MC1R-TUBB3 chimera. ...
Microtubule proteins form a dynamic component of the cytoskeleton, and play key roles in cellular processes, such as vesicular transport, cell motility and mitosis. Expression of microtubule proteins are often dysregulated in cancer. In particular, the microtubule protein βIII-tubulin, encoded by the TUBB3 gene, is aberrantly expressed in a range of epithelial tumours and is associated with drug resistance and aggressive disease. In normal cells, TUBB3 expression is tightly restricted, and is found almost exclusively in neuronal and testicular tissues. Understanding the mechanisms that control TUBB3 expression, both in cancer, mature and developing tissues will help to unravel the basic biology of the protein, its role in cancer, and may ultimately lead to the development of new therapeutic approaches to target this protein. This review is devoted to the transcriptional and posttranscriptional regulation of TUBB3 in normal and cancerous tissue.
... Classically, MC1R is stimulated by alpha melanocyte stimulating hormone (α-MSH) which activates at least two pathways intracellularly: 1) one which raises intracellular cAMP and 2) another which works through mitogen associated protein kinase and is not dependent on elevated cAMP. The MC1R gene can be alternatively spliced at a polyadenylation site that allows for chimeric protein production with TUBB3, as they are adjacent to one another on chromosome 16 (Kolesnichenko 2012; Herraiz et al. 2015). Moreover, UV radiation and other forms of overstimulation of the melanocortin receptor via α-MSH have been found to switch MC1R transcript production over to MC1R-TUBB3; this change may fine-tune the melanocyte response to UV light over the medium to long-term (Dalziel et al. 2011). ...
... MC1R-TUBB3 chimeric protein still binds α-MSH but fails to raise cAMP secondary to intracellular changes in tertiary protein structure. Chimeric gene and protein production may be an adaptive long-term response to overstimulation of MC1R -which results in maintaining certain intracellular pathways (like MAPK noted above) while mitigating cAMP dependent pathways (Herraiz et al. 2015). The upregulation of this chimeric gene may be either causing the pigmentation changes noted in LPPi or is downstream of the overstimulation of melanocytes that have already occurred secondary to the Koebner Phenomenon or some other cause, yet undiscovered. ...
Lichen Planus Pigmentosus inversus (LPPi) is a rare interface and lichenoid dermatitis (ILD) and supposed variant of lichen planus (LP) that presents as well-demarcated brown to grey macules in flexural and intertriginous areas. LPPi is deemed 'inversus' because its anatomical distribution in skin folds is opposite that seen in lichen planus pigmentosus (LPP) whose pigmented lesions arise on sun-exposed skin. Biopsy is required for the clinical diagnosis of all ILDs. Though multiple clinically-oriented studies have reported differences between LPP, LPPi, and LP, few molecular studies have been performed. In this case study, 3 patients, 2 with LPPi and one with LP, provided samples using minimally invasive whole transcriptome analysis using a dermal biomarker patch. This study confirms the involvement of interferon signaling and T-cell activation in LPPi and suggests an expression profile distinct from LP. Specific genes significantly upregulated in LPPi vs LP include an intergenic splice variant of the primary pigmentation determining receptor in humans and dysregulation of genes essential for ceramide synthesis and construction of the cornified envelope. This work expands upon our knowledge of the pathogenesis of LPPi vs LP, and supports the potential use of this technology in the diagnostic clinical setting to mitigate the need for invasive procedures.
... MC1R gene structure, regulation, and activation The human MC1R gene (16q24) was first isolated from melanocytes (Chhajlani and Wikberg, 1992;Mountjoy et al., 1992). Although intergenic and intragenic splicing has been described (Herraiz et al., 2015), the major transcript encodes a 317 amino acid integral membrane protein with the structural characteristics of the GPCRs, including an extracellular N-terminus, with an N-linked glycosylation site, seven transmembrane segments, and an intracellular C-terminal extension, including a key C-terminal palmitoylation site at cysteine 315 (C315) (Chen et al., 2017;García-Borró n et al., 2005;Wolf Horrell et al., 2016) (Figure 1a and b). ...
... As a consequence, in addition to wild-type (WT) MC1R, chimeric MC1ReTUBB3 receptors can be produced in humans (Figure 1c). However, the chimeric proteins produced show a reduced receptor functionality, most notably reduced cell surface expression and an inability to signal through cAMP but a retention of extracellular signal-regulated kinase (ERK) signaling (Dalziel et al., 2011;Herraiz et al., 2015). However, although the mRNAs corresponding to these chimeric MC1ReTUBB3 receptors have been detected, the expression of the chimeric proteins in human skin has yet to be found. ...
The G protein-coupled MC1R is expressed in melanocytes and has a pivotal role in human skin pigmentation, with reduced function in human genetic variants exhibiting a red hair phenotype and increased melanoma predisposition. Beyond its role in pigmentation, MC1R is increasingly recognized as promoting UV-induced DNA damage repair. Consequently, there is mounting interest in targeting MC1R for therapeutic benefit. However, whether MC1R expression is restricted to melanocytes or is more widely expressed remains a matter of debate. In this paper, we review MC1R function and highlight that unbiased analysis suggests that its expression is restricted to melanocytes, granulocytes, and the brain.
... The MC1R gene may exhibit splice variants, giving rise to two forms of intergenic splicing, yielding MC1R-TUBB3 (β-tubulin III) chimera and at least two forms of alternative splicing [13,14]. In all cases, the proteins encoded by the non-canonical mRNAs preserve the general architecture of GPCRs and differ from canonical MC1R for a longer C-terminal extension [14,15]. Once MC1R mRNA is translated, the receptor undergoes post-translational modifications that include oligomerization, N-glycosylation (Asn15, Asn29), palmitoylation (Cys315) and phosphorylation (Thr157, Thr308, Ser316) [11,[16][17][18][19], contributing to receptor structure, localization, trafficking, internalization, desensitization. ...
Melanoma and non-melanoma skin cancers (NMSCs) are the most frequent cancers of the skin in white populations. An increased risk in the development of skin cancers has been associated with the combination of several environmental factors (i.e., ultraviolet exposure) and genetic background, including melanocortin-1 receptor (MC1R) status. In the last few years, advances in the diagnosis of skin cancers provided a great impact on clinical practice. Despite these advances, NMSCs are still the most common malignancy in humans and melanoma still shows a rising incidence and a poor prognosis when diagnosed at an advanced stage. Efforts are required to underlie the genetic and clinical heterogeneity of melanoma and NMSCs, leading to an optimization of the management of affected patients. The clinical implications of the impact of germline MC1R variants in melanoma and NMSCs’ risk, together with the additional risk conferred by somatic mutations in other peculiar genes, as well as the role of MC1R screening in skin cancers’ prevention will be addressed in the current review.
... This behaviour is reminiscent of common and penetrant RHC alleles such as R151C, R160W or D294H. [9,20] We showed previously that homologous desensitization of MC1R-dependent cAMP signalling involves phosphorylation of the C-terminal Thr308 and/or Ser316 residues and binding of ARRB2, which triggers internalization of the receptor-hormone complex. [13,14] The interaction of MC1R and ARRB2 also results in ARRB2 ubiquitylation, a process likely catalysed by the E3 ubiquitin ligase MGRN1, since it is enhanced by MGRN1 overexpression and impaired by siRNA-mediated silencing. ...
The melanocortin 1 receptor (MC1R) is a major determinant of skin pigmentation and sensitivity to ultraviolet radiation. When stimulated by its natural agonists, it promotes the switch from synthesis of poorly photoprotective and lightly colored pheomelanins to production of photoprotective and darker eumelanins. In addition to an unusually high number of single nucleotide polymorphisms, the MC1R is expressed as 3 protein‐coding splice variants. Two transcripts display different 5’ untranslated sequences but yield the same open reading frame corresponding to the canonical 317 aminoacids protein (termed MC1R). An alternative transcript named MC1R‐203 encodes for a 382 amino acids protein of poorly characterized functional properties containing an additional 65 aminoacids C‐terminal extension. Given the known roles of the MC1R C‐terminal extension in forward trafficking, coupling to intracellular effectors and desensitization, the different structure of this domain in MC1R and MC1R‐203 may lead to significant functional alteration(s). We have assessed the functional properties of MC1R‐203, as compared with the canonical MC1R form. We show that unstimulated HBL human melanoma cells express the MC1R‐203 spliceoform, although at much lower levels than canonical MC1R. When expressed in heterologous HEK293 cells, the presence of the 65 aminoacid‐long cytosolic extension immediately after Cys316 in MC1R‐203 did not impair the intracellular stability of the protein, but it interfered with functional coupling to the cAMP cascade and with the ubiquitylation of ARRB2 associated with MC1R desensitization. Conversely, MC1R‐203 retained full capacity to activate ERK1/2 signaling. Accordingly, MC1R‐203 displays biased signaling when expressed in HEK293 cells.
... Calmodulin like skin proteins are already known as markers for keratinocyte differentiation [30]. Interestingly, TUBB3 resides in a very close proximity to the 3′ UTR region of MC1R and intergenic splicing between MC1R and TUBB3 has been reported [8,21]. Interestingly, treatment with the MC1R agonist α-MSH or activation of the stress response kinase p38-MAPK, both key players associated with ultraviolet radiation dermal insult and subsequent skin tanning, resulted in a shift in expression from MC1R in favour of chimeric MC1R-TUBB3 isoforms in cultured melanocytes. ...
... Interestingly, treatment with the MC1R agonist α-MSH or activation of the stress response kinase p38-MAPK, both key players associated with ultraviolet radiation dermal insult and subsequent skin tanning, resulted in a shift in expression from MC1R in favour of chimeric MC1R-TUBB3 isoforms in cultured melanocytes. It has been, thereby, proposed that these chimeric proteins serve to equip melanocytes with novel cellular phenotypes required as part of the pigmentation response [8,21]. On the other hand, CEP152 or centrosomal protein of 152 kDa plays very important role in cell division. ...
Skin pigmentation in human is a complex trait, which varies widely, both within and between human populations. The exact players governing the trait of skin pigmentation remain elusive till date. Various Genome Wide Association Studies (GWAS) have shown the association of different genomic variants with normal human skin pigmentation, often indicating genes with no direct implications in melanin biosynthesis or distribution. Little has been explained in terms of the functionality of the associated Single-Nucleotide Polymorphisms (SNPs) with respect to modulating the skin pigmentation phenotype. In the present study, which, to our knowledge, is the first of its kind, we tried to analyze and prioritize 519 non-coding SNPs and 24 3′UTR SNPs emerging from 14 different human skin pigmentation-related GWAS, primarily using several ENCODE-based web-tools like rSNPBase, RegulomeDB, HaploReg, etc., most of which incorporate experimentally validated evidences in their predictions. Using this comprehensive, in-silico, analytical approach, we successfully prioritized all the pigmentation-associated GWAS-SNPs and tried to annotate pigmentation-related functionality to them, which would pave the way for deeper understanding of the molecular basis of human skin pigmentation variations.
... cAMP was determined using a commercial immunoassay from Arbor Assays (Eisenhower Place, Michigan, USA) [88]. ...
The melanocortin 1 receptor gene (MC1R), a well-established melanoma susceptibility gene, regulates the amount and type of melanin pigments formed within epidermal melanocytes. MC1R variants associated with increased melanoma risk promote the production of photosensitizing pheomelanins as opposed to photoprotective eumelanins. Wild-type (WT) MC1R activates DNA repair and antioxidant defenses in a cAMP-dependent fashion. Since melanoma-associated MC1R variants are hypomorphic in cAMP signaling, these non-pigmentary actions are thought to be defective in MC1R-variant human melanoma cells and epidermal melanocytes, consistent with a higher mutation load in MC1R-variant melanomas. We compared induction of antioxidant enzymes and DNA damage responses in melanocytic cells of defined MC1R genotype. Increased expression of catalase (CAT) and superoxide dismutase (SOD) genes following MC1R activation was cAMP-dependent and required a WT MC1R genotype. Conversely, pretreatment of melanocytic cells with an MC1R agonist before an oxidative challenge with Luperox decreased (i) accumulation of 8-oxo-7,8-dihydro-2′-deoxyguanine, a major product of oxidative DNA damage, (ii) phosphorylation of histone H2AX, a marker of DNA double-strand breaks, and (iii) formation of DNA breaks. These responses were comparable in cells WT for MC1R or harboring hypomorphic MC1R variants without detectable cAMP signaling. In MC1R-variant melanocytic cells, the DNA-protective responses were mediated by AKT. Conversely, in MC1R-WT melanocytic cells, high cAMP production downstream of MC1R blocked AKT activation and was responsible for inducing DNA repair. Accordingly, MC1R activation could promote repair of oxidative DNA damage by a cAMP-dependent pathway downstream of WT receptor, or via AKT in cells of variant MC1R genotype.
