A Rat Brain Bicistronic Gene with an Internal Ribosome Entry Site Codes
for a Phencyclidine-binding Protein with Cytotoxic
Activity |
| |
Authors: | Dongwei Hui Keshava N Kumar Julie R Mach Ashik Srinivasan Ranu Pal Xiaodong Bao Abdulbaki Agbas Georg H?fner Klaus T Wanner Elias K Michaelis |
| |
Institution: | ‡Department of Pharmacology and Toxicology and §Center for Neurobiology and Immunology Research, University of Kansas, Lawrence, Kansas 66047 and the ¶Department Pharmazie-Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität, 81377 München, Germany |
| |
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-dibenzoa,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.A complex of four proteins purified from brain synaptic membranes was shown
to have recognition sites for l-glutamate,
N-methyl-d-aspartate
(NMDA),4 and other
ligands characteristic of NMDA receptors in brain, including binding sites for
the co-agonist glycine, the modulator spermine, the competitive antagonist
(+)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), and the ion
channel inhibitors thienylcyclohexylpiperidine (TCP) and
(+)-5-methyl-10,11-dihydro-5H-dibenzoa,d]cyclohepten-5,10-imine
(MK-801) (1,
2). Reconstitution of the
purified complex into planar lipid bilayer membranes leads to the formation of
channels with four ion conductance levels upon activation by glutamate or NMDA
in the presence of glycine (3).
These conductances differ from either the predominant NMDA-activated
receptor-ion channels of brain neurons or those formed by reconstitution of
the NMDA receptor subunits (4),
but are similar to those described for ion channels in rat spinal cord motor
neurons (5).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). Expression of
GRINA in heterologous cells leads to activation of mitogen-activated
protein kinases (13),
i.e. it may be involved in signal transduction in neurons. Because of
the potential role of GBP and of the associated membrane complex in cell
signaling, there is a need to fully characterize all components of the complex
and reconstitute the intact complex in cells lacking in its expression. The
genes for two other components of the complex have been cloned, those for the
glycine-binding and CPP-binding proteins. But the gene for the fourth subunit
has not yet been cloned.The fourth protein of the complex was identified on SDS-PAGE as an
∼40-kDa protein. To complete the characterization of this complex of
proteins, the cDNA for the fourth subunit was cloned, and a corresponding
genomic sequence in rat genome was identified. The presence of two open
reading frames (ORFs) in the cloned cDNA, the expression of both ORFs in a
single mRNA in brain, and the translation in brain of the two proteins coded
by the cDNA, led to the investigation of the mechanism of translation of both
ORFs. Translation of both ORFs through an internal ribosome entry sequence
(IRES) was identified, as was the need for the co-expression of the two
proteins to create a functional protein, a phencyclidine-binding protein. |
| |
Keywords: | |
|
|