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.     

Binding of [3H]DMCM, a Convulsive Benzodiazepine Ligand, to Rat Brain Membranes: Preliminary Studies

DMCM (methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) produces convulsions in mice and rats, probably by interacting with benzodiazepine (BZ) receptors. Investigation of specific binding of [3H]DMCM to rat hippocampus and cortex revealed polyphasic saturation curves, indicating a high-affinity site (KD = 0.5-0.8 nM) and a site with lower affinity (KD = 3-6 nM). BZ receptor ligands of various chemical classes, but not other agents, displace [3H]DMCM from specific binding sites--indicating that [3H]DMCM binds to BZ receptors in rat brain. The regional distribution of [3H]DMCM binding is complementary to that of the BZ1-selective radioligand [3H]PrCC. Specific binding of [3H]DMCM (0.1 nM) was reduced by gamma-aminobutyric acid (GABA) receptor agonist to approximately 20% of the control value at 37 degrees C in chloride-containing buffers; the reduction was bicuculline methiodide- and RU 5135-sensitive. The effective concentrations of 10 GABA analogues in reducing [3H]DMCM binding correlated closely to published values for their GABA receptor affinity. Specific binding of [3H]DMCM is regulated by unknown factors; e.g. enhanced binding was found by Ag+ treatment of membranes, in the presence of picrotoxinin, or by exposure to ultraviolet light in the presence of flunitrazepam. In conclusion, [3H]DMCM appears to bind to high-affinity brain BZ receptors, although the binding properties are different from those of [3H]flunitrazepam and [3H]PrCC. These differences might relate in part to subclass selectivity and in part to differences in efficacy of DMCM at BZ receptors.     

Effect of peripheral benzodiazepine receptor (PBR/TSPO) ligands on opening of Ca<Superscript>2+</Superscript>-induced pore and phosphorylation of 3.5-kDa polypeptide in rat brain mitochondria

The effect of nanomolar concentrations of PBR/TSPO ligands—Ro 5-4864, PK11195, and PPIX—on Ca²⁺-induced permeability transition pore (PTP) opening in isolated rat brain mitochondria was investigated. PBR/TSPO agonist Ro 5-4864 (100 nM) and endogenous ligand PPIX (1 μM) were shown to stimulate PTP opening, while antagonist PK11195 (100 nM) suppressed this process. Correlation between PBR ligand action on PTP opening and phosphorylation of a 3.5 kDa polypeptide was investigated. In intact brain mitochondria, incorporation of [γ-³²P]ATP into 3.5 kDa peptide was decreased in the presence of Ro 5-4864 and PPIX and increased in the presence of PK11195. At threshold Ca²⁺ concentrations leading to PTP opening, PBR/TSPO ligands were found to stimulate dephosphorylation of the 3.5 kDa peptide. Specific anti-PBR/TSPO antibody prevented both PTP opening and dephosphorylation of the 3.5-kDa peptide. The peptide was identified as subunit c of F_oF₁-ATPase by Western blot using specific anti-subunit c antibody. The results suggest that subunit c of F_oF₁-ATPase could be an additional target for PBR/TSPO ligands action, is subjected to Ca²⁺- and TSPO-dependent phosphorylation/dephosphorylation, and is involved in PTP operation in mitochondria.     

A single histidine in GABAA receptors is essential for benzodiazepine agonist binding.

Benzodiazepines (BZ) modulate neurotransmitter-evoked chloride currents at the gamma-aminobutyric acid type A (GABAA) receptor, the major inhibitory ion channel in the mammalian brain. This receptor is composed of structurally distinct subunits whose numerous molecular variants underlie the observed diversity in the properties of the BZ site. Pharmacologically distinct BZ sites can be recreated by the recombinant coexpression of any one of six alpha subunits, a beta subunit variant, and the gamma 2 subunit. In these receptors the alpha variant determines the affinity for ligand binding of the BZ site. Notably, the alpha 1 and alpha 6 variants impart on alpha chi beta 2 gamma 2 receptors high and negligible affinity, respectively, to BZ ligands with sedative as well as anxiolytic activities. By exchanging domains between the alpha 1 and alpha 6 variants, we show that a portion of the large extracellular domain determines sensitivity toward these ligands. Furthermore, we identify a single histidine residue in the alpha 1 variant, replaced by an arginine in alpha 6, as a major determinant for high affinity binding of BZ agonists. This residue also plays a role in determining high affinity binding for BZ antagonists. Hence, this histidine present in the alpha 1, alpha 2, alpha 3, and alpha 5 subunits appears to be a key residue for the action of clinically used BZ ligands.     

Peripheral-type benzodiazepine receptor: structure and function of a cholesterol-binding protein in steroid and bile acid biosynthesis

Cholesterol transport from the outer to the inner mitochondrial membrane is the rate-determining step in steroid and bile acid biosyntheses. Biochemical, pharmacological and molecular studies have demonstrated that the peripheral-type benzodiazepine receptor (PBR) is a five transmembrane domain mitochondrial protein involved in the regulation of cholesterol transport. PBR gene disruption in Leydig cells completely blocked cholesterol transport into mitochondria and steroid formation, while PBR expression in bacteria, devoid of endogenous PBR and cholesterol, induced cholesterol uptake and transport. Molecular modeling of PBR suggested that cholesterol might cross the membrane through the five helices of the receptor and that synthetic and endogenous ligands might bind to common sites in the cytoplasmic loops. A cholesterol recognition/interaction amino acid consensus (CRAC) sequence in the cytoplasmic carboxy-terminus of the PBR was identified by mutagenesis studies. In vitro reconstitution of PBR into proteoliposomes demonstrated that PBR binds both drug ligands and cholesterol with high affinity. In vivo polymeric forms of PBR were observed and polymer formation was reproduced in vitro, using recombinant PBR protein reconstituted into proteoliposomes, associated with an increase in drug ligand binding and reduction of cholesterol-binding capacity. This suggests that the various polymeric states of PBR might be part of a cycle mediating cholesterol uptake and release into the mitochondria, with PBR functioning as a cholesterol exchanger against steroid product(s) arising from cytochrome P450 action. Taking into account the widespread presence of PBR in many tissues, a more general role of PBR in intracellular cholesterol transport and compartmentalization might be considered.     

Design, synthesis, and structure-activity relationships of novel tetracyclic compounds as peripheral benzodiazepine receptor ligands

The peripheral benzodiazepine receptor (PBR) is pharmacologically distinct from the central benzodiazepine receptor (CBR) and has been identified in a wide range of peripheral tissues as well as in the central nervous system. Although numerous studies have been performed of it, the physiological roles and functions of the PBR are still unclear. In the present study, in exploring new types of ligands for PBR, we found that a new series of compounds having a tetracyclic ring system, which were designed from FGIN-1-27, exhibited high affinities for PBR. We prepared and evaluated them for PBR affinities. The results of binding tests showed that 12e and 12f were the most potent PBR ligands among them (12e: IC(50)=0.44nM, 12f: IC(50)=0.37nM). In this paper, we present the design, synthesis, and structure-activity relationships (SARs) of novel tetracyclic compounds.     

Effect of noise exposure on rat cardiac peripheral benzodiazepine receptors

Noise is an environmental physical agent, which is regarded as a stressful stimulus: impairment and modifications in biological functions are reported, after loud noise exposure, at several levels in human and animal organs and apparatuses, as well as in the endocrine, cardiovascular and nervous system. In the present study equilibrium binding parameters of peripheral benzodiazepine receptors (PBRs) labelled by the specific radioligand [3H]PK 11195, were evaluated in cardiac tissue of rats submitted to 6 or 12 h noise exposure and of rats treated "in vivo" with PBR ligands such as PK 11195, Ro54864, diazepam and then noise-exposed. Results revealed a statistically significant decrease in the maximum number of binding sites (Bmax) of [3H]PK 11195 in atrial membranes of 6 or 12 h noise exposed rats, compared with sham-exposed animals, without any change in the dissociation constant (Kd). The "in vivo" PBR ligand pre-treatment counteracted the noise-induced modifications of PBR density. As PBRs are mainly located on mitochondria we also investigated whether noise exposure can affect the [3H]PK 11195 binding parameters in isolated cardiac mitochondrial fractions. Results indicated a significant Bmax value decrease in right atrial mitochondrial fractions of rats 6 or 12 h noise-exposed. Furthermore, as PBR has been suggested to be a supramolecular complex that might coincide with the not-yet-established structure of the mitochondrial permeability transition (MPT)-pore, the status of the MPT-pore in isolated heart mitochondria was investigated in noise- and sham-exposed rats. The loss of absorbance associated with the calcium-induced MPT-pore opening was greater in mitochondria isolated from hearts of 6 h noise- than those of sham-exposed rats. In conclusion, these findings represent a further instance for PBR density decrease in response to a stressful stimulus, like noise; in addition they revealed that "in vivo" administration of PBR ligands significantly prevents this decrease. Finally, our data also suggest the involvement of MPT in the response of an organism to noise stress.     

PK 11195 attenuates kainic acid-induced seizures and alterations in peripheral-type benzodiazepine receptor (PBR) protein components in the rat brain

Peripheral-type benzodiazepine receptors (PBR) are located in glial cells in the brain and in peripheral tissues. Mitochondria form the primary location for PBR. Functional PBR appear to require at least three components: an isoquinoline binding protein, a voltage-dependent anion channel, and an adenine nucleotide carrier. In the present study, rats received intraperitoneal kainic acid injections, which are known to cause seizures, neurodegeneration, hyperactivity, gliosis, and a fivefold increase in PBR ligand binding density in the hippocampus. In the forebrain of control rats, hippocampal voltage-dependent anion channel and adenine nucleotide carrier abundance was relatively low, while isoquinoline binding protein abundance did not differ between hippocampus and the rest of the forebrain. One week after kainic acid injection, isoquinoline binding protein abundance was increased more than 20-fold in the hippocampal mitochondrial fraction. No significant changes were detected regarding hippocampal voltage-dependent anion channel and adenine nucleotide carrier abundance. Pre-treatment with the isoquinoline PK11195, a specific PBR ligand, attenuated the occurrence of seizures, hyperactivity, and increases in isoquinoline binding protein levels in the hippocampus, which usually follow kainic acid application. These data suggest that isoquinoline binding protein may be involved in these effects of kainic acid injections.     

Synthesis, labeling, and biological evaluation of halogenated 2-quinolinecarboxamides as potential radioligands for the visualization of peripheral benzodiazepine receptors

The previous exploration of the structure-affinity relationships concerning 4-phenyl-2-quinolinecarboxamide peripheral benzodiazepine receptor (PBR) ligands 6 showed as an interesting result the importance of the presence of a chlorine atom in the methylene carbon at position 3 of the quinoline nucleus. The subnanomolar PBR affinity shown by N-benzyl-3-chloromethyl-N-methyl-4-phenyl-2-quinolinecarboxamide (6b) suggested its chlorine atom to be replaced with other halogens in order to optimize the interaction of the quinolinecarboxamide derivatives with PBR and to develop suitable candidates for positron emission tomography (PET) or single photon emission computed tomography (SPECT) studies. The binding studies led to the discovery of fluoromethyl derivative 6a, which showed an IC50 value of 0.11 nM and is, therefore, one of the most potent PBR ligands so far described. Fluoromethyl derivative 6a has been labeled with 11C (t1/2=20.4 min, beta+=99.8%) starting from the corresponding des-methyl precursor (14) using [11C]CH3I in the presence of tetrabutylammonium hydroxide in DMF with a 35-40% radiochemical yield (corrected for decay) and 1.5 Ci/micromol of specific radioactivity. Ex vivo rat biodistribution and inhibition (following intravenous pre-administration of PK11195) studies showed that [11C]6a rapidly and specifically accumulated in PBR-rich tissues such as heart, lung, kidney, spleen, and adrenal, and at a lower level in other peripheral organs and in the brain. The images obtained in mouse with small animal YAP-(S)PET essentially confirmed the result of the ex vivo biodistribution experiments. The biological data suggest that [11C]6a is a promising radioligand for peripheral benzodiazepine receptor PET imaging in vivo.     

TNF-alpha induced PMN apoptosis in whole human blood: Protective effect of SSR180575, a potent and selective peripheral benzodiazepine ligand

The peripheral benzodiazepine receptor (PBR) has been shown to play a key role in the regulation of the mitochondrial process leading to apoptosis. Despite much controversy in the literature on this subject, PBR synthetic ligands (and specifically agonists such as Ro5-4864 and SSR180575) are described as presenting potent anti-apoptotic effect against oxidative stress, TNFα- and tamoxifen-induced apoptosis when the PBR ligand is administrated at a low dose, close to the affinity range of the ligand to its receptor. Such anti-apoptotic activity has already been correlated with a protective effect of PBR ligands against ischemia-reperfusion induced tissue dysfunction.Previously, we had shown that SSR180575 is a specific and high affinity PBR ligand of potential interest in pathological cardiovascular, renal and neurodegenerative indications. Beyond its expression in steroid-producing tissues, heart, liver and kidney, the PBR is also known to be highly expressed in blood cells. In this work, we demonstrate by flow cytometry experiments, that SSR180575, at low concentrations, is able to protect polymorphonuclear leukocytes (PMNs) against TNFα-induced apoptosis in whole blood. Thus, in a new context, SSR180575 again shows potent anti-apoptotic properties. Moreover, TNFα- induced PMN apoptosis appears to be a good surrogate marker for determining SSR180575 blood availability and activity in treated patients.     

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.     

Peripheral-type benzodiazepine receptors in human cerebral cortex, kidney, and colon.

The specific binding of [3H]PK 11195 and [3H]Ro 5-4864 to human cerebral cortex, kidney, and colon membranes was studied in order to determine whether peripheral type benzodiazepine receptors (PBR) characteristics located in human tissues are similar to those located in calf or rat tissues. While [3H]PK 11195 (0.05-10 nM, final concentration) bound with high affinity (KD about 2 nM) to human cerebral cortex, kidney, and colon membranes, yielding maximal numbers of binding sites of 255 +/- 23, 1908 +/- 28, and 1633 +/- 98 fmol/mg protein, respectively, the specific binding of [3H]Ro 5-4864 (1.25-40 nM, final concentration), was barely detectable (nonspecific binding about 90% of the total binding). Furthermore, unlabeled PK 11195 was two orders of magnitude more potent than unlabeled Ro 5-4864 in displacing [3H]PK 11195 specific binding from human cerebral cortex and kidney membranes. These results indicate that PBR binding characteristics located in human tissues are similar (but not identical) to those located in calf tissues, but not to those located in rat tissues.     

Ivermectin and nodulisporic acid receptors in Drosophila melanogaster contain both gamma-aminobutyric acid-gated Rdl and glutamate-gated GluCl alpha chloride channel subunits

35S-labeled derivatives of the insecticides nodulisporic acid and ivermectin were synthesized and demonstrated to bind with high affinity to a population of receptors in Drosophila head membranes that were previously shown to be associated with a glutamate-gated chloride channel. Nodulisporic acid binding was modeled as binding to a single population of receptors. Ivermectin binding was composed of at least two kinetically distinct receptor populations, only one of which was associated with nodulisporic acid binding. The binding of these two ligands was modulated by glutamate, ivermectin, and antagonists of invertebrate gamma-aminobutyric acid (GABA)ergic receptors. Because solubilized nodulisporic acid and ivermectin receptors comigrated as 230-kDa complexes by gel filtration, antisera specific for both the Drosophila glutamate-gated chloride channel subunit GluCl alpha (DmGluCl alpha) and the GABA-gated chloride channel subunit Rdl (DmRdl) proteins were generated and used to examine the possible coassembly of these two subunits within a single receptor complex. DmGluCl alpha antibodies immunoprecipitated all of the ivermectin and nodulisporic acid receptors solubilized by detergent from Drosophila head membranes. DmRdl antibodies also immunoprecipitated all solubilized nodulisporic receptors, but only approximately 70% of the ivermectin receptors. These data suggest that both DmGluCl alpha and DmRdl are components of nodulisporic acid and ivermectin receptors, and that there also exists a distinct class of ivermectin receptors that contains the DmGluCl alpha subunit but not the DmRdl subunit. This co-association of DmGluCl alpha and DmRdl represents the first biochemical and immunological evidence of coassembly of subunits from two different subclasses of ligand-gated ion channel subunits.     

Identification of amino acid residues responsible for the alpha5 subunit binding selectivity of L-655,708, a benzodiazepine binding site ligand at the GABA(A) receptor

L-655,708 is a ligand for the benzodiazepine site of the gamma-aminobutyric acid type A (GABA(A)) receptor that exhibits a 100-fold higher affinity for alpha5-containing receptors compared with alpha1-containing receptors. Molecular biology approaches have been used to determine which residues in the alpha5 subunit are responsible for this selectivity. Two amino acids have been identified, alpha5Thr208 and alpha5Ile215, each of which individually confer approximately 10-fold binding selectivity for the ligand and which together account for the 100-fold higher affinity of this ligand at alpha5-containing receptors. L-655,708 is a partial inverse agonist at the GABA(A) receptor which exhibited no functional selectivity between alpha1- and alpha5-containing receptors and showed no change in efficacy at receptors containing alpha1 subunits where amino acids at both of the sites had been altered to their alpha5 counterparts (alpha1Ser205-Thr,Val212-Ile). In addition to determining the binding selectivity of L-655,708, these amino acid residues also influence the binding affinities of a number of other benzodiazepine (BZ) site ligands. They are thus important elements of the BZ site of the GABA(A) receptor, and further delineate a region just N-terminal to the first transmembrane domain of the receptor alpha subunit that contributes to this binding site.     

A comparative autoradiography study in post mortem whole hemisphere human brain slices taken from Alzheimer patients and age-matched controls using two radiolabelled DAA1106 analogues with high affinity to the peripheral benzodiazepine receptor (PBR) system

The binding of two radiolabelled analogues (N-(5-[125I]Iodo-2-phenoxyphenyl)-N-(2,5-dimethoxybenzyl)acetamide ([125I]desfluoro-DAA1106) and N-(5-[125I]Fluoro-2-phenoxyphenyl)-N-(2-[125I]Iodo-5-methoxybenzyl)acetamide ([125I]desmethoxy-DAA1106) of the peripheral benzodiazepine receptor (PBR) (or TSPO, 18kDa translocator protein) ligand DAA1106 was examined by in vitro autoradiography on human post mortem whole hemisphere brain slices obtained from Alzheimer's disease (AD) patients and age-matched controls. Both [(125)I]desfluoro-IDAA1106 and [(125)I]desmethoxy-IDAA1106 were effectively binding to various brain structures. The binding could be blocked by the unlabelled ligand as well as by other PBR specific ligands. With both radiolabelled compounds, the binding showed regional inhomogeneity and the specific binding values proved to be the highest in the hippocampus, temporal and parietal cortex, the basal ganglia and thalamus in the AD brains. Compared with age-matched control brains, specific binding in several brain structures (temporal and parietal lobes, thalamus and white matter) in Alzheimer brains was significantly higher, indicating that the radioligands can effectively label-activated microglia and the up-regulated PBR/TSPO system in AD. Complementary immunohistochemical studies demonstrated reactive microglia activation in the AD brain tissue and indicated that increased ligand binding coincides with increased regional microglia activation due to neuroinflammation. These investigations yield further support to the PBR/TSPO binding capacity of DAA1106 in human brain tissue, demonstrate the effective usefulness of its radio-iodinated analogues as imaging biomarkers in post mortem human studies, and indicate that its radiolabelled analogues, labelled with short half-time bioisotopes, can serve as prospective in vivo imaging biomarkers of activated microglia and the up-regulated PBR/TSPO system in the human brain.     

The peripheral-type benzodiazepine receptor is functionally linked to Leydig cell steroidogenesis

Testicular mitochondria were previously shown to contain an abundance of peripheral-type benzodiazepine recognition site(s)/receptor(s) (PBR). We have previously purified, cloned, and expressed an Mr 18,000 PBR protein (Antkiewicz-Michaluk, Mukhin, A. G., Guidotti, A., and Krueger, K. E. (1988) J. Biol. Chem. 263, 17317-17321; (Sprengel, R., Werner, P., Seeburg, P. H., Mukhin, A. G., Santi, M. R., Grayson, D. R., Guidotti, A., and Krueger, K. E. (1989) J. Biol. Chem. 264, 20415-20421); and in this report, we present evidence that PBR are functionally linked to Leydig cell steroid biosynthesis. A spectrum of nine different ligands covering a range of over 4 orders of magnitude in their affinities for PBR were tested for their potencies to modulate steroidogenesis in the MA-10 mouse Leydig tumor cell line. The Ki for inhibition of [3H]1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide binding and the EC50 for steroid biosynthesis for this series of compounds showed a correlation coefficient of r = 0.95. The most potent ligands stimulated steroid production by approximately 4-fold in these cells. This stimulation was not inhibited by cycloheximide, unlike human chorionic gonadotropin- or cyclic AMP-activated steroidogenesis. The action of PBR ligands was not additive to stimulation by human chorionic gonadotropin or cyclic AMP, but was additive to that of epidermal growth factor, another regulator of MA-10 Leydig cell steroidogenesis. Moreover, PBR ligands stimulated, in a dose-dependent manner, pregnenolone biosynthesis by isolated mitochondria when supplied with exogenous cholesterol. This effect was not observed with mitoplasts (mitochondria devoid of the outer membrane). Cytochrome P-450 side chain cleavage activity, as measured by metabolism of (22R)-hydroxycholesterol, was not affected by PBR ligands in intact cells. Similar results were also obtained with purified rat Leydig cells. In conclusion, PBR are implicated in the acute stimulation of Leydig cell steroidogenesis possibly by mediating the entry, distribution, and/or availability of cholesterol within mitochondria.     

Cloning, expression, and functional characterization of the substrate binding subunit of rat type II iodothyronine 5'-deiodinase

Type II iodothyronine 5'-deiodinase catalyzes the bioactivation of thyroid hormone in the brain. In astrocytes, this approximately 200-kDa, membrane-bound enzyme is composed of at least one p29 subunit, an approximately 60-kDa, cAMP-induced activation protein, and one or more unidentified catalytic subunit(s). Recently, an artificial type II-like selenodeiodinase was engineered by fusing two independent cDNAs together; however, no native type II selenodeiodinase polypeptide is translated in the brain or brown adipose tissue of rats. These data suggest that the native type II 5'-deiodinase in rat brain is unrelated to this artificial selenoprotein. In this report, we describe the cloning of the 29-kDa subunit (p29) of type II 5'-deiodinase from a lambdazapII cDNA library prepared from cAMP-induced astrocytes. The 3.3-kilobase (kb) cDNA encodes an approximately 30-kDa, 277-amino acid long, hydrophobic protein lacking selenocysteine. Northern blot analysis showed that a 3.5-kb p29 mRNA was present in tissues showing type II 5'-deiodinase activity such as brain and cAMP-stimulated astrocytes. Domain-specific, anti-p29 antibodies specifically immunoprecipitated enzyme activity. Overexpression of exogenous p29 or a green fluorescence protein (GFP)-tagged p29 fusion protein led to a >100-fold increase in deiodinating activity in cAMP-stimulated astrocytes, and the increased activity was specifically immunoprecipitated by anti-GFP antibodies. Steady-state reaction kinetics of the enzyme in GFP-tagged p29-expressing astrocytes are identical to those of the native enzyme in brain. Direct injection of replication-deficient Ad5-p29(GFP) virus particles into the cerebral cortex of neonatal rats leads to a approximately 2-fold increase in brain type II 5'-deiodinating activity. These data show 1) that the 3.3-kb p29 cDNA encodes an essential subunit of rat type II iodothyronine 5'-deiodinase and 2) identify the first non-selenocysteine containing subunit of the deiodinase family of enzymes.