M. Garnier et al., IN-VITRO RECONSTITUTION OF A FUNCTIONAL PERIPHERAL-TYPE BENZODIAZEPINE RECEPTOR FROM MOUSE LEYDIG TUMOR-CELLS, Molecular pharmacology, 45(2), 1994, pp. 201-211
The peripheral-type benzodiazepine receptor (PBR) was identified and c
haracterized by its high affinity for two distinct classes of compound
s, the benzodiazepines (BZs) and the isoquinolines (IQs). An M(r) 18,0
00 IQ-binding protein has been identified as the PBR. In this report w
e isolated and sequenced a 626-base pair cDNA, specifying an open read
ing frame of 169 amino acid residues with a predicted molecular weight
of 18,843, from MA-10 mouse tumor Leydig cells [i.e., mouse periphera
l-type benzodiazepine receptor (mPBR)]. Expression of mPBR cDNA in sim
ian virus 40-transformed 3T3 fibroblasts resulted in an increase in th
e density of both BZ and IQ binding sites. To examine whether the incr
eased drug binding was due to the M(r), 18,000 PBR protein alone or to
other constitutively expressed components of the receptor, an in vitr
o system was developed using recombinant mPBR protein. The mPBR cDNA w
as inserted in the pMAL-c2 vector downstream from the malE gene, which
encodes maltose-binding protein (MBP). Transfection of the recombinan
t pMAL-c2 in Escherichia coli provided high levels of expression of th
e MBP-mPBR fusion protein. Purified MBP-mPBR recombinant fusion protei
n incorporated into liposomes, but not MBP alone, was able to bind IQs
but not BZs. Addition of MA-10 mitochondrial extracts to the liposome
s resulted in the restoration of BZ binding. The protein responsible f
or this effect was then purified and identified as the M(r) 34,000 vol
tage-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 th
e 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-depen
dent anion channel subunits.