Molecular variation and evolution of the tyrosine kinase domains of insulin receptor IRa and IRb genes in Cyprinidae

SCIENCE CHINA Life Sciences • RESEARCH PAPERS • July 2011 Vol.54 No.7: 626–633 doi: 10.1007/s11427-011-4189-3 Molecular variation and evolution of the tyrosine kinase domains of insulin receptor IRa and IRb genes in Cyprinidae KONG XiangHui1,2, WANG XuZhen2 & HE ShunPing2* 2 1 College of Life Science, Henan Normal University, Xinxiang 453007, China; Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China Received December 3, 2010; accepted May 30, 2011; published online June 23, 2011 The insulin receptor (IR) gene plays an important role in regulating cell growth, differentiation and development. In the present study, DNA sequences of insulin receptor genes, IRa and IRb, were amplified and sequenced from 37 representative species of the Cyprinidae and from five outgroup species from non-cyprinid Cypriniformes. Based on coding sequences (CDS) of tyrosine kinase regions of IRa and IRb, molecular evolution and phylogenetic relationships were analyzed to better understand the characteristics of IR gene divergence in the family Cyprinidae. IRa and IRb were clustered into one lineage in the gene tree of the IR gene family, reconstructed using the unweighted pair group method with arithmetic mean (UPGMA). IRa and IRb have evolved into distinct genes after IR gene duplication in Cyprinidae. For each gene, molecular evolution analyses showed that there was no significant difference among different groups in the reconstructed maximum parsimony (MP) tree of Cyprinidae; IRa and IRb have been subjected to similar evolutionary pressure among different lineages. Although the amino acid sequences of IRa and IRb tyrosine kinase regions were highly conserved, our analyses showed that there were clear sequence variations between the tyrosine kinase regions of IRa and IRb proteins. This indicates that IRa and IRb proteins might play different roles in the insulin signaling pathway. insulin receptor gene, tyrosine kinase domain, Cyprinidae Citation: Kong X H, Wang X Z, He S P. Molecular variation and evolution of the tyrosine kinase domains of insulin receptor IRa and IRb genes in Cyprinidae. Sci China Life Sci, 2011, 54: 626–633, doi: 10.1007/s11427-011-4189-3 Insulin and insulin-like growth factor play important roles in growth regulation, cell differentiation and metabolism [1–6]. Their actions are mediated by the corresponding member of the insulin receptor family. The insulin receptor family comprises the insulin receptor (IR), insulin-like growth factors receptors (IGFRs) and the insulin receptor-related receptor (IRR). IR and IGFRs are cross-membrane glycoproteins with similar structures [7,8]. The mature IR comprises two α and two β subunits, which form heterologous α2β2 tetramers connected via disulfide bonds. The IR protein is composed of a binding region in its α-subunit and a transmembrane domain and tyrosine kinase region in its β-sub*Corresponding author (email: ) © The Author(s) 2011. This article is published with open access at Springerlink.com unit [7,8]. The tyrosine kinase activity of the β-subunit can be inhibited by the α-subunit. When insulin binds to the α-subunit, the β-subunit kinase activity inhibitor can be released by phosphorylation and the α-subunit changes its conformation, thereby enhancing tyrosine kinase activity [9]. A combination of insulin and IR results in a cascade of phosphorylation to active downstream regulatory signals. Insulin receptors (IR/IGFR/IRR) belong to the tyrosine kinase receptor family, the kinase activity of which is located in the tyrosine kinase region. Study of the molecular variation and evolution of the tyrosine kinase region of the IR gene would help in the understanding of IR gene divergence and of different IR roles in insulin signaling. In fish, sequence characteristics and expression patterns life.scichina.com www.springer.com/scp Kong X H, et al. Sci China Life Sci of IR and IGFR genes have been studied in rainbow trout (Oncorhynchus mykiss) [10], coho salmon (O. kisutch) [11], turbot (Scophthalmus maximus) [10,12], Pacific chum salmon (O. keta) [13] and chinook salmon (O. tshawytscha) [14]. These IR genes contain a conserved tyrosine kinase domain of about 400 bp [11]. IR, IGF-IR and IGF-IIR have also been cloned in Danio rerio [1,15]. These studies indicate duplications of IR and IGFR genes in fish. Toyoshima et al. [1] cloned and characterized IRa and IRb from zebrafish. They also studied their expression patterns and found overlapping functions of the two genes during zebrafish embryogenesis. However, these studies are limited to an individual species or to a few species of fish. It would be of great interest to investigate IR gene divergence among closely related fish in certain families by analyzing molecular variation and evolution of tyrosine kinase coding sequences (CDSs) of IR genes. The family Cyprinidae is the largest family of freshwater fish and contains 2010 species in about 210 genera [16]. Cyprinids have a wide distribution and are highly diverse with obvious differences in size, food habits and niches among species. Therefore, they are considered as an ideal group to study functional gene divergence. In this study, molecular variation and evolution of the tyrosine kinase domain of IRa and IRb were analyzed in the family Cyprinidae. This study aimed to identify variation of the deduced amino acid sequence of the tyrosine kinase domain between IRa and IRb proteins within the family Cyprinidae and to Table 1 627 July (2011) Vol.54 No.7 understand the molecular evolution and divergence of IRa and IRb. 1 Materials and methods 1.1 Sample collection The nucleotide sequences of IRa and IRb were determined in 37 representative species from the family Cyprinidae and in 5 species from non-cyprinid Cypriniformes. All specimens were from the collections of the Freshwater Fish Museum of the Institute of Hydrobiology, Chinese Academy of Sciences. The collection location, deposition identification and GenBank accession number of all species are listed in Table 1. Muscle or fin tissue preserved in 95% ethanol was used to extract genome DNA. 1.2 Primer design Based on the conserved regions of D. rerio IRa (AF400271.1; NM_001142672.1) and IRb (AF400272.1; NM_001123229), primers were designed and optimized to amplify Ira (forward primer 5′-ACGGTYAAYGAGTCKGCCAGYCT-3′, reverse primer 5′-CYTCRGCCACCATGCAGTTC-3′) and IRb (forward primer 5′-GMGHGAGAGRATHGAGTTC-3′, reverse primer 5′-CAGCCACCATGCAGTTYCTDG 3′). Species of cyprinid and outgroup taxa investigated in this study Species Sampling location Voucher Culter alburnus Elopichthys bambusa Megalobrama amblycephala Aristichthys nobilis Squaliobarbus curriculus Ctenopharyngodon idellus Pseudobrama simoni Mylopharyngodon piceus Hemiculter leucisculus Ochetobius elongates Xenocypris argentea Aphyocypris chinensis Opsariichthys bidens Saurogobio gracilicaudatus Saurogobio dabryi Gobiocypris rarus Pseudorasbora parva Coreius heterodon He (...truncated)