ISSN 10681620, Russian Journal of Bioorganic Chemistry, 2016, Vol. 42, No. 4, pp. 449–452. © Pleiades Publishing, Ltd., 2016. Original Russian Text © D.M. Shayahmetova, E.S. Zhevlenev, A.A. Mozhaev, I.E. Deyev, A.G. Petrenko, 2016, published in Bioorganicheskaya Khimiya, 2016, Vol. 42, No. 4, pp. 496–500.
Genetic Link between IRRReceptor and Ly6/PLAUR Protein1 D. M. Shayahmetova2, E. S. Zhevlenev, A. A. Mozhaev, I. E. Deyev, and A. G. Petrenko Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. MiklukhoMaklaya 16/10, Moscow, 117997 Russia Received December 25, 2015; in final form, February 5, 2016
Abstract—Receptor tyrosine kinase IRR is a pHsensor that able to be activated by alkaline extracellular media. IRR is a close homologue of the insulin receptor and insulinlike growth factor receptor but no endog enous ligands of a peptide or protein nature have been found for IRR. IRR expression was detected in certain populations of kidney cells, stomach, pancreas and also in parts of the sympathetic and cholinergic neurons. We have previously identified behavioral disorders in mice lacking IRR. In this study we performed a com parative analysis of wildtype mice brain transcriptomes and IRRknockout mice brain transcriptomes using method of deep sequencing. The most significant difference (twofold increase in expression) was observed for Ly6/PLAUR protein transcript. It is known that this protein is able to stimulate nicotinic acetylcholine receptor desensibilization. These results suggest that behavioral characteristics of mice lacking IRR are caused by the functional link between IRR and Ly6/PLAUR in cholinergic neurons of the brain. Keywords: receptor tyrosine kinase IRR, sequencing, littermates (homozygous mouse one generation), acetylcho line receptor DOI: 10.1134/S1068162016040166 1
INTRODUCTION The insulin receptorrelated receptor (IRR) is an orphan receptor tyrosine kinase that belongs to the mini family of the insulin receptor which also includes the insulinlike growth factor receptor the and insulin receptor [1]. IRR was discovered by cloning in 1989 and, since then, the attempts to find its protein/pep tide ligands have been unsuccessful [2, 3]. In our search for the endogenous agonist of IRR, we found that IRR, in contrast to its close homologs, can be activated by extracellular media with pH > 7.9. Bio chemical analysis showed that the activation of IRR by hydroxyl ion has characteristic features of ligand receptor interactions determined by the structure of its extracellular domain [4, 5]. IRR is expressed in certain populations of kidney, stomach, pancreas cells and also in the nervous sys tem, specifically in subpopulations parts of sympa thetic and cholinergic neurons [6–9]. In general, this distribution pattern supports the hypothesis of a key role of IRR as a sensor of a mildly alkaline medium, since these tissues (except for those of the nervous sys tem) are characterized by significant local pH varia tion, including weakly alkaline values [10]. Experi ments in vivo with IRRknockout mice showed that IRR is involved in the removal of excess bases by the Abbreviations: IRR, insulin receptorrelated receptor; GPI, glyco sylphosphatidylinositol; SPF, sterile zone keeping animals. 1 The article was translated by the authors. 2 Corresponding author: phone: +7 (495) 3354177; fax: +7 (495) 3350812; email:
[email protected].
kidneys that is important to maintaining the acidbase balance in the body [5, 11, 12]. IRRknockout mice exhibited a phenotype that might be related to the function of the nervous system. As a result of the IRR gene knockout, changes in the social and aggressivedefensive behavior the mice were observed [13]. Due to the fact that substantial devia tions from neutral pH values for the nervous system tissue are not known, the pHsensing function of IRR in the regulation of animal behavior is unclear. To identify the specific molecular and cellular mecha nisms of the IRR function in the nervous system, we conducted a comparative analysis of the brain tran scriptomes of wild type and IRR knockout mice by NGS and found a statistically significant difference in the expression of Ly6/PLAUR/Lynx2 protein. RESULTS AND DISCUSSION In order to identify the role of IRR in the central nervous system homeostasis regulation, we conducted a comparative analysis of the transcriptome of wild type mice brain and IRRknockout mice brain using NGS. The analysis was performed with three pairs of homozygous littermate wildtype and IRRknockout mice. RNA was isolated from brain samples obtained by separation of cortex from the rest of the brain. Then, cDNA was prepared and carried out by NGS on the Illumina platform. About 7.7 million reads per sample for IRRknockout mice and 5.1 million reads for wildtype mice were obtained.
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As the version of the reference genome was used Mus musculus Ensembl, mm10. Read mapping was carried out with TopHat2 program [14]. During the analysis of differentially expressed genes all types of RNA (rRNA, miRNA, snRNA, snoRNA, misc RNA, mt_tRNA, pseudogenes), except mRNA, were masked. The differential expression analysis was per formed by Rpackage DESeq [15]. Genes were fil tered when the total number of reads in which was less than 20, and the genes for which reads present less than in three samples in the comparison group. Signif icant changes in expression were considered with p value
