title: Functional Characterization of MC1R-TUBB3 Intergenic Splice Variants of the Human Melanocortin 1 Receptor.
abstract: 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.
authors.affiliation: Department of Biochemistry, Molecular Biology and Immunology, School of Medicine, University of Murcia, Campus de Espinardo, Murcia and IMIB-Arrixaca, El Palmar, Murcia, Spain.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Herraiz
authors.affiliation: Department of Biochemistry, Molecular Biology and Immunology, School of Medicine, University of Murcia, Campus de Espinardo, Murcia and IMIB-Arrixaca, El Palmar, Murcia, Spain.
authors.forename: Conchi
authors.initials: C
authors.lastname: Olivares
authors.affiliation: Department of Biochemistry, Molecular Biology and Immunology, School of Medicine, University of Murcia, Campus de Espinardo, Murcia and IMIB-Arrixaca, El Palmar, Murcia, Spain.
authors.forename: Maria
authors.initials: M
authors.lastname: Castejón-Griñán
authors.affiliation: Department of Biochemistry, Molecular Biology and Immunology, School of Medicine, University of Murcia, Campus de Espinardo, Murcia and IMIB-Arrixaca, El Palmar, Murcia, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Abrisqueta
authors.affiliation: Department of Biochemistry, Molecular Biology and Immunology, School of Medicine, University of Murcia, Campus de Espinardo, Murcia and IMIB-Arrixaca, El Palmar, Murcia, Spain.
authors.forename: Celia
authors.initials: C
authors.lastname: Jiménez-Cervantes
authors.affiliation: Department of Biochemistry, Molecular Biology and Immunology, School of Medicine, University of Murcia, Campus de Espinardo, Murcia and IMIB-Arrixaca, El Palmar, Murcia, Spain.
authors.forename: José Carlos
authors.initials: JC
authors.lastname: García-Borrón
mesh_terms: D000242:Cyclic AMP / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D008544:Melanocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D050506:Mutant Chimeric Proteins / Q000235:genetics* / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D021381:Protein Transport; D012326:RNA Splicing; D044102:Receptor, Melanocortin, Type 1 / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D014404:Tubulin / Q000235:genetics* / Q000378:metabolism; D014466:Ultraviolet Rays; D000521:alpha-MSH / Q000378:metabolism / Q000494:pharmacology
chemical_list: D050506:Mutant Chimeric Proteins; D020033:Protein Isoforms; D044102:Receptor, Melanocortin, Type 1; C578986:TUBB1 protein, human; D014404:Tubulin; D000521:alpha-MSH; D000242:Cyclic AMP; D048049:Extracellular Signal-Regulated MAP Kinases