Functional diversification of sea urchin ABCC1 (MRP1) by alternative splicing

Blastopore view of sea urchin early gastrulae

Blastopore view of sea urchin early gastrulae

TitleFunctional diversification of sea urchin ABCC1 (MRP1) by alternative splicing
Publication TypeJournal Article
Year of Publication2016
AuthorsGokirmak T., Campanale J.P, Reitzel A.M, Shipp L.E, Moy G.W, Hamdoun A.
JournalAmerican Journal of Physiology-Cell Physiology
Date Published2016/06
Type of ArticleArticle
ISBN Number0363-6143
Accession NumberWOS:000384745400008
KeywordsATP-binding cassette transporters; binding cassette transporter; c-4; cancer; development; drug; efflux pumps; export pump; expression; gene family; known as ABCC1); leukotriene; mast-cells; multidrug resistance protein 1 (also; multidrug-resistance protein-1; protein evolution; Sea urchin; strongylocentrotus-purpuratus

The multidrug resistance protein (MRP) family encodes a diverse repertoire of ATP-binding cassette (ABC) transporters with multiple roles in development, disease, and homeostasis. Understanding MRP evolution is central to unraveling their roles in these diverse processes. Sea urchins occupy an important phylogenetic position for understanding the evolution of vertebrate proteins and have been an important invertebrate model system for study of ABC transporters. We used phylogenetic analyses to examine the evolution of MRP transporters and functional approaches to identify functional forms of sea urchin MRP1 (also known as SpABCC1). SpABCC1, the only MRP homolog in sea urchins, is co-orthologous to human MRP1, MRP3, and MRP6 (ABCC1, ABCC3, and ABCC6) transporters. However, efflux assays revealed that alternative splicing of exon 22, a region critical for substrate interactions, could diversify functions of sea urchin MRP1. Phylogenetic comparisons also indicate that while MRP1, MRP3, and MRP6 transporters potentially arose from a single transporter in basal deuterostomes, alternative splicing appears to have been the major mode of functional diversification in invertebrates, while duplication may have served a more important role in vertebrates. These results provide a deeper understanding of the evolutionary origins of MRP transporters and the potential mechanisms used to diversify their functions in different groups of animals.

Short TitleAm. J. Physiol.-Cell Physiol.

The results of this study extend and validate the heterologous expression approach we have adapted for analysis of sea urchin ABC transporters. These assays are important, because they reveal the transporters that could be responsible for observed activities and fingerprint the relevant suspects for analysis by more laborious immunochemical methods. Large overexpression has been used to ensure that the vast majority of the observed efflux activity comes from the recombinant protein, which is essential for characterization of substrate selectivity.

Here, we showed that for localization studies the protein can be reproducibly titrated to levels that increase corresponding transport activity only twofold and still be functionally imaged by routine confocal microscopy (Fig. 4B), albeit with use of high-sensitivity detectors. More importantly, these experiments indicated that localization of both forms of sea urchin ABCC1 could be both apical and bilateral, depending on developmental stage and independent of expression level. This dual location could be an additional mechanism for diversification of function in both protection and signaling.

Finally, an important step was to demonstrate the expression of functional human MRP1. An interesting observation was that while sea urchin protein exhibited both apical and basolateral efflux activity, the human protein only appeared to act at the basolateral membrane, possibly indicating that the differences in localization of sea urchin and human proteins are likely to be inherent features of the transporters themselves. Collectively, the results indicate that the sea urchin is an important system in which to understand the evolution of MRP proteins.

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