Genomic organization of the human GRIK2 gene and evidence for multiple splicing variants q Alessandro Barbon, Ivan Vallini, Sergio Barlati * Division of Biology and Genetics, Department of Biomedical Sciences and Biotechnologies, Brescia University Medical School, via Valsabbina 19, 25123 Brescia, Italy Received 9 January 2001; received in revised form 2 July 2001; accepted 11 July 2001 Received by M. D’Urso Abstract Fast excitatory transmission in the vertebrate central nervous system is mediated mainly by l-glutamate. Here we present the genomic organization of the human GRIK2 gene, which codes for the kainate GluR6 receptor subunit, deduced from sequence data present in the public databases and analyzed by bioinformatic tools. By similarity search using the human GluR6 cDNA sequence against non-redundant databases, we found three positive entries (AP002528, AP002529, and AP002530 deposited by Hirakawa et al., 2000) which are part of a BAC contig of about 1 Mb spanning region 6q21. The GRIK2 gene was found to be split into 17 exons, covering about 670 kb of the region. The availability of the data on the genomic organization allowed the study of GRIK2 gene expression by RT-PCR analysis which was performed on human teratocarcinoma cell cultures (NT2) and on mRNA obtained from human hippocampus (Clontech). The study gives evidence for several different splicing variants in addition to the previously cloned human GluR6 cDNA (ID: U16126). The splicing mechanism leading to the different isoforms involves exons 11, 12 and 16. The mRNA containing exon 16 at the 3 0 end is the homolog to the mouse GluR6-2. The translation of this mRNA would code for a different intracellular C-terminus, as compared to that coded by the known human isoform. The newly identify isoform is the predominant form expressed in human teratocarcinoma NT2 cells. All the newly identified mRNAs isoforms are expressed in NT2 cells and in human hippocampus mRNA at variable levels and would be responsible for the production of five different putative GluR6 receptor subunits, some differing in the C-terminal domains (mouse homolog) and some lacking specific transmembrane domains. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Glutamate receptors; Bioinformatic; Alternative splicing; Reverse transcription-polymerase chain reaction; Sequencing; Human teratocarcinoma cells; Human hippocampus 1. Introduction Glutamate receptors (GluRs) mediate most of the excita- tory neurotransmission in the central nervous system; in addition they are involved in plastic changes in synaptic transmission and are thought to play a role in neurological disorders, such as stroke, ischemia or progressing neurode- generative disorders (for review see Dingledine et al., 1999; Ozawa et al., 1998). The GluRs are divided into two distinct groups, called metabotropic and ionotropic GluRs. The metabotropic receptors (mGluRs) are coupled to GTP-binding proteins and regulate the production of intracellular messengers. The ionotropic receptors (iGluRs), that assemble together forming ion channels, are further divided into three groups on the basis of agonist specificities: a-amino-3-hydroxy-5- methyl-4-isoxazolepropionate (AMPA), kainate and N- methyl-d-aspartate (NMDA) receptor channels. Mammals express four AMPA (GluR1, GluR2, GluR3, and GluR4), five kainate (GluR5, GluR6, GluR7, KA1, and KA2) and six NMDA receptor subunits (NR1, NR2A, NR2B, NR2C, NR2D and NR3A). In addition, two d subu- nits (d1 and d2) have also been found but their function is presently unknown (Lomeli et al., 1993). The molecular diversity of iGluRs is further increased by variants due to alternative splicing and RNA editing (Bigge, 1999). All iGluR subunits are approximately 900 aa long and share three transmembrane domains (TM1, TM3, and TM4) and a re-entrant membrane loop (M2) on the cytoplasmic Gene 274 (2001) 187–197 0378-1119/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S0378-1119(01)00611-4 www.elsevier.com/locate/gene Abbreviations: dsRNA, double-strand RNA; GluR, glutamate receptor (protein); GRIK, glutamate receptor ionotropic kainate (gene); NT2, tera- tocarcinoma cell line; RT, reverse transcription; TM, transmembrane domain; TSS, transcriptional start site; UTR, untranslated region q Sequences submitted with Accession numbers: AJ301608, AJ301609, and AJ301610. * Corresponding author. Tel.: 139-030-3717244; fax: 139-030- 3701157. E-mail address: barlati@med.unibs.it (S. Barlati).