A Rat Brain Bicistronic Gene with an Internal Ribosome Entry Site Codes for a Phencyclidine-binding Protein with Cytotoxic Activity

Dongwei Hui,Keshava N Kumar,Julie R Mach,Ashik Srinivasan,Ranu Pal,Xiaodong Bao,Abdulbaki Agbas,Georg Höfner,Klaus T Wanner,Elias K Michaelis

doi:10.1074/jbc.m807063200

Dongwei Hui, Keshava N Kumar + Show 8 more

Open Access

https://doi.org/10.1074/jbc.m807063200

Copy DOI

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.

Highlights

The genes for three of the proteins in this complex have been cloned and expressed in heterologous cells (6 –10)
The gene GRINA for the glutamate-binding protein (GBP) subunit was identified as part of a “learning and memory” module of genes expressed in the entorhinal cortex of the mammalian brain [11], and as the gene responsible for mental retardation and epilepsy in infants with a gene duplication in chromosome 8q24.3 [12]
No amplification products were detected if Reverse Transcriptase (RT) was not included in the reaction. These results indicated that mRNA with the sequence of the 1.3-kb cDNA insert, including ORF1, ORF2, and the inter-open reading frames (ORFs) sequence, was present in brain as intact mRNA

Summary

EXPERIMENTAL PROCEDURES

Purification of the ϳ40-kDa Protein and Raising of Polyclonal Antibodies—Synaptic membranes were prepared from two rat brains [14, 15], and membrane proteins were solubilized in 25 ml of Buffer A (1% CHAPS, 0.5% n-octylglucopyranoside, 10% glycerol, 10 mM potassium phosphate buffer, pH 7.4, plus protease inhibitors) [7] and subjected to centrifugation (100,000 ϫ g for 1 h). The oligonucleotides 5Ј-AGCTTTTCTTGGCATTCCGGTACTGTTGGTAAA-3Ј and 5Ј-GATCTTTACCAACAGTACCGGAATGCCAAGAAA-3Ј were annealed, and 10 ng of this double-stranded DNA was ligated to 400 ng of pGL3 pre-digested with BglI/HindIII (removal of SV40 promoter) To this vector, the sequence 5Ј-CTTTACCAACAGTACCGGAATGCCAAG-3Ј, predicted to form a thermodynamically stable stem-loop hairpin (hp) in mRNA when combined with the palindromic sequence 3ЈGAAATGGTTGTCATGGCCTTACGGTTC-5Ј in pGL3 vector, was introduced 5 bases upstream of the initiation codon of firefly luciferase. Noncapped mRNA containing firefly luciferase/inter-ORF/ Gaussia luciferase was synthesized using vector SMMRL (20 ␮g) cut with SspI, purified, and introduced as template (5 ␮g) in RiboMAXTM large scale RNA production system, SP6 (Promega). Cell Transfection with Cloned cDNA, Reporter Vectors, and Noncapped mRNA and Measurement of Luciferase Activity— CHO and SK-N-SH cells were used for protein expression studies. Cell viability was determined at either 48 h (CHO cells) or 72 h (SK-N-SH cells) post-transfection by measuring (Celltiter 96 kit, Promega) the reduction of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) [22]

RESULTS AND DISCUSSION

The inhibitory concentration for

Systematic Name