title: A rat brain bicistronic gene with an internal ribosome entry site codes for a phencyclidine-binding protein with cytotoxic activity.
abstract: The cloning and characterization of the gene for the fourth subunit of a glutamate-binding protein complex in rat brain synaptic membranes are described. The cloned rat brain cDNA contained two open reading frames (ORFs) encoding 8.9- (PRO1) and 9.5-kDa (PRO2) proteins. The cDNA sequence matched contiguous genomic DNA sequences in rat chromosome 17. Both ORFs were expressed within the structure of a single brain mRNA and antibodies against unique sequences in PRO1- and PRO2-labeled brain neurons in situ, indicative of bicistronic gene expression. Dicistronic vectors in which ORF1 and ORF2 were substituted by either two different fluorescent proteins or two luciferases indicated concurrent, yet independent translation of the two ORFs. Transfection with noncapped mRNA led to cap-independent translation of only ORF2 through an internal ribosome entry sequence preceding ORF2. In vitro or cell expression of the cloned cDNA led to the formation of multimeric protein complexes containing both PRO1 and PRO2. These complexes had low affinity (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801)-sensitive phencyclidine-binding sites. Overexpression of PRO1 and PRO2 in CHO cells, but not neuroblastoma cells, caused cell death within 24-48 h. The cytotoxicity was blocked by concurrent treatment with MK-801 or by two tetrahydroisoquinolines that bind to phencyclidine sites in neuronal membranes. Co-expression of two of the other subunits of the protein complex together with PRO1/PRO2 abrogated the cytotoxic effect without altering PRO1/PRO2 protein levels. Thus, this rare mammalian bicistronic gene coded for two tightly interacting brain proteins forming a low affinity phencyclidine-binding entity in a synaptic membrane complex.
authors.affiliation: Department of Pharmacology and Toxicology, Center for Neurobiology and Immunology Research, University of Kansas, Lawrence, Kansas 66047, USA.
authors.forename: Dongwei
authors.initials: D
authors.lastname: Hui
authors.forename: Keshava N
authors.initials: KN
authors.lastname: Kumar
authors.forename: Julie R
authors.initials: JR
authors.lastname: Mach
authors.forename: Ashik
authors.initials: A
authors.lastname: Srinivasan
authors.forename: Ranu
authors.initials: R
authors.lastname: Pal
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Bao
authors.forename: Abdulbaki
authors.initials: A
authors.lastname: Agbas
authors.forename: Georg
authors.initials: G
authors.lastname: Höfner
authors.forename: Klaus T
authors.initials: KT
authors.lastname: Wanner
authors.forename: Elias K
authors.initials: EK
authors.lastname: Michaelis
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016923:Cell Death; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D003603:Cytotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D030561:Databases, Nucleic Acid; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016366:Open Reading Frames / Q000235:genetics; D010622:Phencyclidine / Q000378:metabolism*; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000235:genetics; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012270:Ribosomes / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics
chemical_list: D002352:Carrier Proteins; D003603:Cytotoxins; D018076:DNA, Complementary; D009419:Nerve Tissue Proteins; C539169:PRO1 protein, rat; C539170:PRO2 protein, rat; D021122:Protein Subunits; D010622:Phencyclidine