Molecular analysis of alternative transcripts of equine AXL receptor tyrosine kinase gene

  • Park, Jeong-Woong ;
  • Song, Ki-Duk ;
  • Kim, Nam Young ;
  • Choi, Jae-Young ;
  • Hong, Seul A ;
  • Oh, Jin Hyeog ;
  • Kim, Si Won ;
  • Lee, Jeong Hyo ;
  • Park, Tae Sub ;
  • Kim, Jin-Kyoo ;
  • Kim, Jong Geun ;
  • Cho, Byung-Wook
  • Received : 2017.05.25
  • Accepted : 2017.07.28
  • Published : 2017.10.01


Objective: Since athletic performance is a most importance trait in horses, most research focused on physiological and physical studies of horse athletic abilities. In contrast, the molecular analysis as well as the regulatory pathway studies remain insufficient for evaluation and prediction of horse athletic abilities. In our previous study, we identified AXL receptor tyrosine kinase (AXL) gene which was expressed as alternative spliced isoforms in skeletal muscle during exercise. In the present study, we validated two AXL alternative splicing transcripts (named as AXLa for long form and AXLb for short form) in equine skeletal muscle to gain insight(s) into the role of each alternative transcript during exercise. Methods: We validated two isoforms of AXL transcripts in horse tissues by reverse transcriptase polymerase chain reaction (RT-PCR), and then cloned the transcripts to confirm the alternative locus and its sequences. Additionally, we examined the expression patterns of AXLa and AXLb transcripts in horse tissues by quantitative RT-PCR (qRT-PCR). Results: Both of AXLa and AXLb transcripts were expressed in horse skeletal muscle and the expression levels were significantly increased after exercise. The sequencing analysis showed that there was an alternative splicing event at exon 11 between AXLa and AXLb transcripts. 3-dimentional (3D) prediction of the alternative protein structures revealed that the structural distance of the connective region between fibronectin type 3 (FN3) and immunoglobin (Ig) domain was different between two alternative isoforms. Conclusion: It is assumed that the expression patterns of AXLa and AXLb transcripts would be involved in regulation of exercise-induced stress in horse muscle possibly through an $NF-{\kappa}B$ signaling pathway. Further study is necessary to uncover biological function(s) and significance of the alternative splicing isoforms in race horse skeletal muscle.


Horse;AXL Receptor Tyrosine Kinase;Alternative Splicing;Athletic Performance;Muscle;RNA-Sequence


  1. Cho HW, Shin S, Park JW, et al. Molecular characterization and expression analysis of the peroxisome proliferator activated receptor delta ($PPAR{\delta}$) gene before and after exercise in horse. Asian-Australas J Anim 2015;28:697-702.
  2. Kallberg M, Wang H, Wang S, et al. Template-based protein structure modeling using the RaptorX web server. Nat Protoc 2012;7:1511-22.
  3. Madhusudhan MS, Marti-Renom MA, Eswar N, et al. Comparative protein structure modeling. The Proteomics Protocols Handbook, Totowa, NJ: Humana Press; 2005. pp. 831-60.
  4. Rose PW, Beran B, Bi C, et al. The RCSB protein data bank: redesigned web site and web services. Nucleic Acids Res 2011;39(Suppl 1):D392-D401.
  5. MacKerell AD, Banavali N, Foloppe N. Development and current status of the CHARMM force field for nucleic acids. Biopolymers 2000;56:257-65.<257::AID-BIP10029>3.0.CO;2-W
  6. Holland SJ, Powell MJ, Franci C, et al. Multiple roles for the receptor tyrosine kinase axl in tumor formation. Cancer Res 2005;65:9294-303.
  7. Hasanbasic I, Cuerquis J, Varnum B, Blostein MD. Intracellular signaling pathways involved in Gas6-AXL-mediated survival of endothelial cells. Am J Physiol-Heart C, 2004;287:H1207-H13.
  8. Ji LL, Gomez-Cabrera MC, Steinhafel N, Vina J. Acute exercise activates nuclear factor (NF)-${\kappa}B$ signaling pathway in rat skeletal muscle. FASEB J 2004;18:1499-506.
  9. Llinas, M. Metal-polypeptide interactions: The conformational state of iron proteins. In: Metal Bonding in Proteins. Berlin, Heidelberg: Springer; 1973. p. 135-220.
  10. Gu J, Orr N, Park SD, et al. A genome scan for positive selection in thoroughbred horses. PloSOne 2009;4:e5767.
  11. Park KD, Park J, Ko J, et al. Whole transcriptome analyses of six thoroughbred horses before and after exercise using RNA-Seq. BMC Genomics 2012;13:473.
  12. O'Bryan JP, Frye RA, Cogswell PC, et al. AXL, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase. Mol Cell Biol 1991;11:5016-31.
  13. Sainaghi PP, Castello L, Bergamasco L, et al. Gas6 induces proliferation in prostate carcinoma cell lines expressing the AXLreceptor. J Cell Physiol 2005;204:36-44.
  14. McCloskey P, Fridell YW, Attar E, et al. GAS6 mediates adhesion of cells expressing the receptor tyrosine kinase AXL. J Biol Chem 1997; 272:23285-91.
  15. Tai KY, Shieh YS, Lee CS, Shiah SG, Wu CW. AXL promotes cell invasion by inducing MMP-9 activity through activation of NF-${\kappa}B$ and Brg-1. Oncogene 2008;27:4044-55.
  16. Cunningham BA, Hemperly JJ, Murray BA, et al. Neural cell adhesion molecule: structure, immunoglobulin-like domains, cell surface modulation, and alternative RNA splicing. Science 1987;236:799-807.
  17. Fischer EH, Charbonneau H, Tonks NK. Protein tyrosine phosphatases: a diverse family of intracellular and transmembrane enzymes. Science 1991;253:401-7.
  18. Varnum BC, Young C, Elliott G, et al. AXL receptor tyrosine kinase stimulated by the vitamin K-dependent protein encoded by growtharrest- specific gene 6. Nature 1995;373:623.
  19. Stitt TN, Conn G, Goret M, et al. The anticoagulation factor protein S and its relative, Gas6, are ligands for the Tyro 3/Axl family of receptor tyrosine kinases. Cell 1995;80:661-70.
  20. Sasaki T, Knyazev PG, Clout NJ, et al. Structural basis for Gas6-Axl signalling. EMBO J 2006;25:80-7.
  21. Goruppi S, Ruaro E, Varnum B, Schneider C. Requirement of phosphatidylinositol 3-kinase-dependent pathway and Src for Gas6-Axl mitogenic and survival activities in NIH 3T3 fibroblasts. Mol Cell Biol 1997;17:4442-53.
  22. Hasanbasic I, Cuerquis J, Varnum B, Blostein MD. Intracellular signaling pathways involved in Gas6-Axl-mediated survival of endothelial cells. Am J Physiol-Heart C 2004;287:H1207-13.
  23. Paccez JD, Vogelsang M, Parker MI, Zerbini LF. The receptor tyrosine kinase AXL in cancer: biological functions and therapeutic implications. Int J Cancer 2014;134:1024-33.
  24. Rankin EB, Fuh KC, Castellini L, et al. Direct regulation of GAS6/AXL signaling by HIF promotes renal metastasis through SRC and MET. Proc Natl Acad Sci USA 2014;111:13373-8.
  25. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the $2^{-{\Delta}{\Delta}CT}$method. Methods 2001;25:402-8.
  26. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987;4:406-25.


Supported by : Rural Development Administration