In vitro reconstitution of a functional peripheral-type benzodiazepine receptor from mouse Leydig tumor cells.
The peripheral-type benzodiazepine receptor (PBR) was identified and characterized by its high affinity for two distinct classes of compounds, the benzodiazepines (BZs) and the isoquinolines (IQs). An M(r) 18,000 IQ-binding protein has been identified as the PBR. In this report we isolated and sequenced a 626-base pair cDNA, specifying an open reading frame of 169 amino acid residues with a predicted molecular weight of 18,843, from MA-10 mouse tumor Leydig cells [i.e., mouse peripheral-type benzodiazepine receptor (mPBR)]. Expression of mPBR cDNA in simian virus 40-transformed 3T3 fibroblasts resulted in an increase in the density of both BZ and IQ binding sites. To examine whether the increased drug binding was due to the M(r) 18,000 PBR protein alone or to other constitutively expressed components of the receptor, an in vitro system was developed using recombinant mPBR protein. The mPBR cDNA was inserted in the pMAL-c2 vector downstream from the malE gene, which encodes maltose-binding protein (MBP). Transfection of the recombinant pMAL-c2 in Escherichia coli provided high levels of expression of the MBP-mPBR fusion protein. Purified MBP-mPBR recombinant fusion protein incorporated into liposomes, but not MBP alone, was able to bind IQs but not BZs. Addition of MA-10 mitochondrial extracts to the liposomes resulted in the restoration of BZ binding. The protein responsible for this effect was then purified and identified as the M(r) 34,000 voltage-dependent anion channel protein, which by itself does not express any BZ and IQ binding. These results provide strong evidence that PBR is not a single protein receptor but a multimeric complex in which the IQ binding site is on the M(r) 18,000 subunit and expression of the BZ binding site requires both the M(r) 18,000 and 34,000 voltage-dependent anion channel subunits.
Garnier
M
M
Department of Cell Biology, Georgetown University Medical Center, Washington, DC 20007.
Dimchev
A B
AB
Boujrad
N
N
Price
J M
JM
Musto
N A
NA
Papadopoulos
V
V
D016475:3T3 Cells; D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001569:Benzodiazepines / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins; D007546:Isoquinolines / Q000378:metabolism*; D007984:Leydig Cell Tumor / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D051379:Mice; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D016133:Polymerase Chain Reaction; D011963:Receptors, GABA-A / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D014407:Tumor Cells, Cultured
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D007546:Isoquinolines; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011963:Receptors, GABA-A; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D001569:Benzodiazepines