Purification and characterization of a protein associated with peripheral-type benzodiazepine binding sites

The photoaffinity ligand [3H]PK 14105 was utilized to modify covalently peripheral-type benzodiazepine binding sites in rat adrenal mitochondrial preparations. The photolabeled membrane preparations were then solubilized in 1% digitonin and the detergent-soluble extracts subjected to fractionation by ion-exchange chromatography and reversed-phase high performance liquid chromatography. This scheme resulted in the purification of the putative binding site protein for PK 14105 which we have entitled PKBS. Purified preparations of PKBS exhibited a single band with a Mr of approximately 17,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver-staining or autoradiographic detection. Additional criteria examining the purity of PKBS preparations were provided by radioiodination with Bolton-Hunter reagent, amino acid analysis, gas-phase sequencing, and reversed-phase chromatography suggesting that this protein was purified to apparent homogeneity. These results demonstrate that a protein associated with peripheral-type benzodiazepine recognition sites has been isolated thus facilitating more direct studies on the structure of this receptor and on the role of these binding sites in mediating responses elicited by benzodiazepines acting at these sites.     

Hormonal regulation of peripheral-type benzodiazepine receptors

Central benzodiazepine (BZ) receptors are located only in the central nervous system and mediate the clinical effects obtained by various BZs. In addition, there is another receptor that binds BZs with different drug specificities, which is located mainly on the outer mitochondrial membrane of various peripheral tissues. Peripheral BZ receptors (PBR) are composed of three subunits: an isoquinoline binding site, a voltage-dependent anion channel, and an adenine nucleotide carrier, with molecular weights of 18, 32, and 30 kDa, respectively. Complementary DNA of the isoquinoline binding subunit has been cloned in rat, calf, and human. The major role of PBR is in the regulation of steroid biosynthesis. Various PBR ligands stimulate the conversion of cholesterol into pregnenolone and the production of steroid hormones. The naturally occurring diazepam-binding inhibitor stimulates in vivo steroidogenesis via binding to PBR. In the female, PBR density is increased in rat and human ovary proportional with greater cell maturation and differentiation. In the male, testosterone modulates PBR density in the genital tract. These results show the strong relationship between PBR and the endocrine system.     

Role of the peripheral-type benzodiazepine receptor and the polypeptide diazepam binding inhibitor in steroidogenesis

Steroidogenesis begins with the metabolism of cholesterol to pregnenolone by the inner mitochondrial membrane cytochrome P450 side-chain cleavage (P450scc) enzyme. The rate of steroid formation, however, depends on the rate of (i) cholesterol transport from intracellular stores to the inner mitochondrial membrane and (ii) loading of P450scc with cholesterol. We demonstrated that a key element in the regulation of cholesterol transport is the mitochondrial peripheral-type benzodiazepine receptor (PBR) and that the presence of the polypeptide diazepam binding inhibitor (DBI) was vital for steroidogenesis. We also showed that DBI, as the endogenous PBR ligand, stimulates cholesterol transport. In addition, DBI directly promotes loading of cholesterol to P450scc. We review herein our studies on the structure, function, topography and hormonal regulation of PBR and DBI in steroidogenic cells. Based on these data we propose a model where the interaction of DBI with PBR, at the outer/inner membrane contact sites, is the signal transducer of hormone-stimulated and constitutive steroidogenesis at the mitochondrial level. Hormone-induced changes in PBR microenvironment/structure regulate the affinity of the receptor. PBR ligand binding to a higher affinity receptor results in increased cholesterol transport. In addition, hormone-induced release (processing?) of a 30,000 M_W DBI-immunoreactive protein from the inner mitochondrial membrane may result to the intramitochondrial production of DBI which directly stimulates loading of P450scc with cholesterol. Thus, in vivo, hormonal activation of these two mechanisms results in efficient cholesterol delivery and utilization and thus high levels of steroid synthesis.     

Cloning and expression of a pharmacologically unique bovine peripheral-type benzodiazepine receptor isoquinoline binding protein.

High affinity binding of isoquinolines, such as PK 11195, is a conserved feature of peripheral-type benzodiazepine receptors (PBR) across species. However, species differences in PBR ligand binding have been described based on the affinity for N1-alkyl-1,4-benzodiazepines, such as Ro5-4864. Ro5-4864 binds with high affinity to the rat receptor but has low affinity for the bovine PBR. Photolabeling with an isoquinoline ligand, [3H]PK 14105, identifies a 17-kDa protein, the PBR isoquinoline binding protein (PBR/IBP), in both species. To further elucidate the role of the PBR/IBP in determining PBR benzodiazepine and isoquinoline binding characteristics, the bovine PBR/IBP was cloned and expressed. Using a cDNA encoding a rat PBR/IBP to screen a fetal bovine adrenal cDNA library, a bovine cDNA encoding a polypeptide of 169 residues was cloned. The bovine and rat PBR/IBPs had similar hydropathy profiles exhibiting five potential transmembrane domains. Transfecting the cloned bovine PBR/IBP cDNA into COS-7 cells resulted in an 11-fold increase in the density of high affinity [3H]PK 11195 binding sites which had only low affinity for Ro5-4864. Expression of the bovine PBR/IBP yields a receptor which is pharmacologically distinct from both endogenous COS-7 PBR and the rat PBR based on the affinity for several N1-alkyl-1,4-benzodiazepine ligands. These results suggest the PBR/IBP is the minimal functional component required for PBR ligand binding characteristics and the different protein sequences account for the species differences in PBR benzodiazepine ligand binding.     

Barbiturate and benzodiazepine modulation of GABA receptor binding and function

The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) acts primarily on receptors that increase chloride permeability in postsynaptic neurons. These receptors are defined by sensitivity to the agonist muscimol and the antagonist bicuculline, and are also subject to indirect allosteric inhibition by picrotoxin-like convulsants and enhancement by the clinically important drugs, the benzodiazepines and the barbiturates. All of these drugs modulate GABA-receptor regulated chloride channels at the cellular level assayed by electrophysiological or radioactive ion tracer techniques. Specific receptor sites for GABA, benzodiazepines, picrotoxin/convulsants, and barbiturates can be assayed in vitro by radioactive ligand binding. Mutual chloride-dependent allosteric interactions between the four receptor sites indicate that they are all coupled in the same membrane macromolecular complex. Indirect effects of barbiturates on the other three binding sites define a pharmacologically specific, stereospecific receptor. All of the activities can be solubilized in the mild detergent 3-[(3-cholamidopropyl)-dimethylammonio]propane sulfonate (CHAPS) and co-purify as a single protein complex.     

Covalent modification of GABAA receptor isoforms by a diazepam analogue provides evidence for a novel benzodiazepine binding site that prevents modulation by these drugs

Classical benzodiazepines, for example diazepam, interact with alpha(x)beta(2)gamma(2) GABA(A) receptors, x = 1, 2, 3, 5. Little is known about effects of alpha subunits on the structure of the binding pocket. We studied here the interaction of the covalently reacting diazepam analog 7-Isothiocyanato-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (NCS compound) with alpha(1)H101Cbeta(2)gamma(2) and with receptors containing the homologous mutation, alpha(2)H101Cbeta(2)gamma(2), alpha(3)H126Cbeta(2)gamma(2) and alpha(5)H105Cbeta(2)gamma(2). This comparison was extended to alpha(6)R100Cbeta(2)gamma(2) receptors as this mutation conveys to these receptors high affinity towards classical benzodiazepines. The interaction was studied at the ligand binding level and at the functional level using electrophysiological techniques. Results indicate that the geometry of alpha(6)R100Cbeta(2)gamma(2) enables best interaction with NCS compound, followed by alpha(3)H126Cbeta(2)gamma(2), alpha(1)H101Cbeta(2)gamma(2) and alpha(2)H101Cbeta(2)gamma(2), while alpha(5)H105Cbeta(2)gamma(2) receptors show little interaction. Our results allow conclusions about the relative apposition of alpha(1)H101 and homologous positions in alpha(2), alpha(3), alpha(5) and alpha(6) with the position occupied by -Cl in diazepam. During this study we found evidence for the presence of a novel site for benzodiazepines that prevents modulation of GABA(A) receptors via the classical benzodiazepine site. The novel site potentially contributes to the high degree of safety to some of these drugs. Our results indicate that this site may be located at the alpha/beta subunit interface pseudo-symmetrically to the site for classical benzodiazepines located at the alpha/gamma interface.     

Selective binding and controlled release of anticancer drugs by polyanionic cyclodextrins

The binding stoichiometry, binding constants, and inclusion mode of some water-soluble negatively charged cyclodextrin derivatives, i.e. heptakis-[6-deoxy-6-(3-sulfanylpropanoic acid)]-β-cyclodextrin(H1), heptakis-[6-deoxy-6-(2-sulfanylacetic acid)]-β-cyclodextrin(H2), mono-[6-deoxy-6-(3-sulfanylpropanoic acid)]-β-cyclodextrin (H3) and mono-[6-deoxy-6-(2-sulfanylacetic acid)]-β-cyclodextrin (H4), with three anticancer drugs, i.e. irinotecan hydrochloride; topotecan hydrochloride; doxorubicin hydrochloride, were investigated by means of ¹H NMR, UV–Vis spectroscopy, mass spectra and 2D NMR. Polyanionic cyclodextrins H1-H2 showed the significantly high binding abilities of up to 2.6?×?10⁴–2.0?×?10⁵ M^?1 towards the selected anticancer drugs, which were nearly 50–1000 times higher than the corresponding Ks values of native β-cyclodextrin. In addition, these polyanionic cyclodextrins also showed the pH-controlled release behaviors. That is, the anticancer drugs could be efficiently encapsulated in the cyclodextrin cavity at a pH value similar to that of serum but sufficiently released at an endosomal pH value of a cancer cell, which would make these cyclodextrin derivatives the potential carriers for anticancer drugs.     

The inhibition of GABA-stimulated benzodiazepine binding by a convulsant benzodiazepine

The binding of ³H-diazepam to benzodiazepine receptors of brain was studied in washed and pre-frozen preparations. The GABA enhancement of ³H-diazepam binding was found to be inhibited by a convulsant benzodiazepine, Ro 5-3663.     

Binding activities of thyroxine binding globulin versus thyroxine binding prealbumin in rat sera: differential modulation by thyroid hormone ligands, oleic acid and pharmacological drugs

We use gel equilibration and electrophoretic techniques to compare the binding properties of thyroxine binding globulin and thyroxine binding prealbumin in rat sera. The evidence indicates that TBG bears the serum lowest capacity highest affinity sites for thyroxine (T4) and triiodothyronine (T3) (Ka1 greater than or equal to 10(9) M-1) as well as weaker saturable T3 sites (Ka2 approximately 10(8) M-1). TBPA bears for T4 only Ka2 approximately 10(8) M-1 sites and for T3 only Ka approximately 10(6) M-1 sites. Consistent with these parameters are the specific responses of TBG and TBPA binding activities to varying serum concentrations of T4, T3, oleic acid, the drugs diphenylhydantoin or salicylate. The primary attack of these compounds is aimed at TBG. Small T4, oleate or DPH doses chase the TBG-bound T4 to TBPA, high doses of T4 or oleate but not of DPH inhibiting the T4 binding to both proteins. In the T3-serum interactions, all tested compounds displace the TBG-bound hormone without chasing it to TBPA. The high reactivity of TBG sites designates the protein as crucially involved in modulating the free vs bound serum levels of T4 and T3 against physiological or pathological variations of binding competitors.     

Differential modulation by muscimol of [3H] flunitrazepam binding to benzodiazepine binding site subtypes

The effects of the GABA agonist, muscimol on [³H]flunitrazepam binding were examined in cerebellum and hippocampus regions proposed to contain different populations of benzodiazepine binding site subtypes. Quantitative analysis was made of the contribution of different components of [³H]flunitrazepam binding by utilising the selective affinities of propyl β-carboline-3-carboxylate for these sites. The influence of muscimol on each of these components was determined and the results provide clear evidence that GABA receptors interact with only some subtypes of benzodiazepine binding sites; for example, whilst the cerebellar site and the low affinity hippocampal site are influenced, the high affinity site in hippocampus appears to be quite unaffected.     

In vivo and in vitro peripheral-type benzodiazepine receptor polymerization: functional significance in drug ligand and cholesterol binding

Peripheral-type benzodiazepine receptor (PBR) is an 18 kDa high-affinity drug ligand and cholesterol binding protein involved in various cell functions. Antisera for distinct PBR areas identified immunoreactive proteins of 18, 40, and 56 kDa and occasionally 72, 90, and 110 kDa in testicular Leydig and breast cancer cells. These sizes may correspond to PBR polymers and correlated to the levels of reactive oxygen species. Treatment of Leydig cells with human chorionic gonadotropin rapidly induced free radical, PBR polymer, and steroid formation. UV photoirradiation generates ROS species, which increased the size of intramembraneous particles of recombinant PBR reconstituted into proteoliposomes consistent with polymer formation, determined both by SDS-PAGE and by freeze-fracture electron microscopy. Spectroscopic analysis revealed the formation of dityrosines as the covalent cross-linker between PBR monomers. Moreover, photoirradiation increased PK 11195 drug ligand binding and reduced cholesterol binding capacity of proteoliposomes. Further addition of PK 11195 drug ligand to polymers increased the rate of cholesterol binding. These data indicate that reactive oxygen species induce in vivo and in vitro the formation of covalent PBR polymers. We propose that the PBR polymer might be the functional unit responsible for ligand-activated cholesterol binding and that PBR polymerization is a dynamic process modulating the function of this receptor in cholesterol transport and other cell-specific PBR-mediated functions.     

Reduced diazepam binding following chronic benzodiazepine treatment.

Specific ³H-diazepam binding was measured in rat cortex after 7–10 days of twice daily injection of a large dose of flurazepam. Compared to cortex taken from saline treated controls, there was a statistically significant decrease of about 15% in the maximum binding capacity (B_max). There was also a change in the dissociation constant (K_D) from 5.27 to 8.80 nM. Repeated washing of the tissue sample before the binding assay showed that the B_max was truly decreased in the treated animals, but the change in K_D was probably an artifact due to residual flurazepam remaining in the tissue that interfered with the binding assay. It is concluded that chronic benzodiazepine treatment caused an apparent decrease in the number of specific binding sites.     

Selective modulation of band 4.1 binding to erythrocyte membranes by protein kinase C

We have studied the effects of band 4.1 phosphorylation on its association with red cell inside-out vesicles stripped of all peripheral proteins. Band 4.1 bound to these vesicles in a saturable manner, and binding was characterized by a linear Scatchard plot with an apparent Kd of 1-2 x 10(-7) M. Phosphorylation of band 4.1 by purified protein kinase C reduced its ability to bind to membranes, resulting in a reduction in the apparent binding capacity of the membrane by 60-70% but little or no change in the apparent Kd of binding. By contrast, phosphorylation of band 4.1 by cAMP-dependent kinase had no effect on membrane binding. Digestion of the stripped inside-out vesicles with trypsin cleaved 100% of the cytoplasmic domain of band 3 but had little or no effect on glycophorin. Binding of band 4.1 to these digested vesicles was reduced by 70%. Phosphorylation of band 4.1 by protein kinase C had no effect on its binding to the digested vesicles, suggesting that the cytoplasmic domain of band 3 contained the phosphorylation-sensitive binding sites. This was confirmed by direct measurement of band 4.1 binding to the purified cytoplasmic domain of band 3. Phosphorylation of band 4.1 by protein kinase C reduced its binding to the purified 43-kDa domain by as much as 90%, while phosphorylation by cAMP-dependent kinase was without effect. These results show a selective effect of protein kinase C phosphorylation on the binding of band 4.1 to one of its membrane receptors, band 3, and suggest a mechanism whereby one of the key red cell-skeletal membrane associations may be modulated.     

The bovine peripheral-type benzodiazepine receptor: a receptor with low affinity for benzodiazepines.

The density of bovine peripheral-type benzodiazepine receptors (PBR) in four tissues was highest in adrenal cortex. The adrenal cortex PBR cofractionated with a mitochondrial membrane marker enzyme and could be solubilized with intact ligand binding properties using digitonin. The membrane bound and soluble mitochondrial receptors were pharmacologically characterized and showed the rank order of potency to inhibit [3H]PK 11195 binding was PK 11195 greater than protoporphyrin IX greater than benzodiazepines (clonazepam, diazepam, or Ro5-4864). [3H]PK 11195 binding to bovine adrenal mitochondria was unaffected by diethylpyrocarbonate, a histidine residue modifying reagent that decreased binding to rat liver mitochondria by 70%. [3H]PK 14105 photolabeled the bovine PBR and the Mr was estimated under nondenaturing (200 kDa) and denaturing (17 kDa) conditions. These results demonstrate the bovine peripheral-type benzodiazepine receptor is pharmacologically and biochemically distinct from the rat receptor, but the receptor component photolabeled by an isoquinoline ligand has a similar molecular weight.     

Inhibitors of peripheral-type benzodiazepine receptors present in human urine and plasma ultrafiltrates

Several endogenous substances that inhibit central-type benzodiazepine (BZD) receptor binding have recently been identified. We have found that ultrafiltrates of human uremic plasma, normal plasma, and urine contain competitive inhibitors of peripheral-type benzodiazepine receptors. Using urine as source, we have partially purified a peripheral-type BZD receptor inhibitor(s) by adsorption to and selective elution from small octadecyl-silane (Sep-pak) columns and thin layer chromatography. The inhibitor has a 125-fold greater affinity for peripheral-type than central-type BZD receptors and has been purified 8000-fold from urine.     

Molecular cloning and chromosomal localization of a human peripheral-type benzodiazepine receptor.

The sequencing of endopeptidase-generated peptides from the peripheral binding site (PBS) for benzodiazepines, purified from a Chinese hamster ovary (CHO) cell line, produced internal sequence information, and confirmed and extended the NH2-terminal PBS sequence that we previously reported. Since the sequences were highly similar to the corresponding rat PBS sequences, we investigated whether they were also conserved in human PBS. Scatchard analysis of [3H]PK11195 (a derivative of isoquinoline carboxamide) binding and photoaffinity labeling with [3H]PK14105 (a nitrophenyl derivative of PK11195) revealed that CHO PBS and human PBS are closely related. Furthermore a rabbit antiserum raised against three peptides synthesized on the basis of the CHO PBS sequence immunoprecipitate the solubilized U937 PBS and also recognize the human protein in an immunoblot analysis. Based on these results, we screened a U937 cell cDNA library with four oligonucleotide probes derived from the CHO sequence. Two of the probes hybridized with several clones that we isolated and sequenced. One of these, h-pPBS11, is 831 nucleotides and contains a full-length representation of human PBS mRNA. The amino acid sequence of human PBS deduced from the cDNA is 79% identical to that reported for rat PBS, however, human PBS contains two cysteines while rat PBS is characterized by the absence of this amino acid. Using the cDNA of human PBS as a probe, the PBS gene was located in the 22q13.3 band of the human genome.