Cholesterol transport via ABCA1: New insights from solid-phase binding assay

It is now well established that the ATP-binding cassette transporter A1 (ABCA1) plays a pivotal role in HDL metabolism, reverse cholesterol transport and net efflux of cellular cholesterol and phospholipids. We aimed to resolve some uncertainties related to the putative function of ABCA1 as a mediator of lipid transport by using a methodology developed in the laboratory to isolate a protein and study its interactions with other compounds. ABCA1 was tagged with the 1D4 peptide at the C terminus and expressed in human HEK 293 cells. Preliminary experiments showed that the tag modified neither the protein expression/localization within the cells nor the ability of ABCA1 to promote cholesterol cellular efflux to apolipoprotein A-I. ABCA1-1D4 was then purified and reconstituted in liposomes. ABCA1 displayed an ATPase activity in phospholipid liposomes that was significantly decreased by cholesterol. Finally, interactions with either cholesterol or apolipoprotein A-I were assessed by binding experiments with protein immobilized on an immunoaffinity matrix. Solid-phase binding assays showed no direct binding of cholesterol or apolipoprotein A-I to ABCA1. Overall, our data support the hypothesis that ABCA1 is able to mediate the transport of cholesterol from cells without direct interaction and that apo A-I primarily binds to membrane surface or accessory protein(s).     

Functional expression of rat ABCG2 on the luminal side of brain capillaries and its enhancement by astrocyte-derived soluble factor(s)

The purpose of the present study was to clarify the expression, transport properties and regulation of ATP-binding cassette G2 (ABCG2) transporter at the rat blood-brain barrier (BBB). The rat homologue of ABCG2 (rABCG2) was cloned from rat brain capillary fraction. In rABCG2-transfected HEK293 cells, rABCG2 was detected as a glycoprotein complex bridged by disulfide bonds, possibly a homodimer. The protein transported mitoxantrone and BODIPY-prazosin. In rat brain capillary fraction, rABCG2 protein was also detected as a glycosylated and disulfide-linked complex. Immunohistochemical analysis revealed that rABCG2 was localized mainly on the luminal side of rat brain capillaries, suggesting that rABCG2 is involved in brain-to-blood efflux transport. For the regulation study, conditionally immortalized rat brain capillary endothelial (TR-BBB13), astrocyte (TR-AST4) and pericyte (TR-PCT1) cell lines were used as an in vitro BBB model. Following treatment of TR-BBB13 cells with conditioned medium of TR-AST4 cells, the Ko143 (an ABCG2-specific inhibitor)-sensitive transport activity and rABCG2 mRNA level were significantly increased, whereas conditioned medium of TR-PCT1 cells had no effect. These results suggest that rat brain capillaries express functional rABCG2 protein and that the transport activity of the protein is up-regulated by astrocyte-derived soluble factor(s) concomitantly with the induction of rABCG2 mRNA.     

Possibility of increasing cholesterol efflux by adiponectin and its receptors through the ATP binding cassette transporter A1 in HEK293T cells

A decrease in adiponectin secretion leads to the early stage of atherosclerosis. Discoidal high-density lipoproteins (HDL) accept the cholesterol that effluxes from cells expressing the ATP binding cassette transporter A1 (ABCA1) in the first step of reverse cholesterol transport (RCT). Recently, a new therapeutic strategy involving reconstituted (r)HDL has been shown to enhance RCT. Therefore, we hypothesized that adiponectin may increase the efflux associated with ABCA1 and also enhance rHDL-induced efflux in human embryonic kidney 293 (HEK293T) cells. We transfected adiponectin receptor 1 and 2 (AdipoR1 and AdipoR2) cDNA into cells. The transfected cells were labeled with [³H]cholesterol following cholesterol loading with or without adiponectin for 24 h. The levels of cholesterol efflux were analyzed using a liquid scintillation counter. Treatment with adiponectin was associated with significantly higher levels of efflux in AdipoR1- and AdipoR2-transfected cells. Interestingly, rHDL-induced cholesterol efflux was enhanced in the presence of adiponectin. The down-regulation of adiponectin receptors using short-hairpin RNA decreased rHDL-induced cholesterol efflux with the down-regulation of ABCA1. In summary, adiponectin and its receptors increased cholesterol efflux and also enhanced rHDL-induced efflux at least partially through an ABCA1 pathway. These results suggest that adiponectin may enhance the RCT system and induce an anti-atherogenic effect.     

Serum amyloid A generates high density lipoprotein with cellular lipid in an ABCA1- or ABCA7-dependent manner

Serum amyloid A (SAA) is an amphiphilic helical protein that is found associated with plasma HDL in various pathological conditions, such as acute or chronic inflammation. Cellular lipid release and generation of HDL by this protein were investigated, in comparison with the reactions by apolipoprotein A-I (apoA-I) and several types of cells that appear with various specific profiles of cholesterol and phospholipid release. SAA mediated cellular lipid release from these cells with the same profile as apoA-I. Upregulation of cellular ABCA1 protein by liver X receptor/retinoid X receptor agonists resulted in an increase of cellular lipid release by apoA-I and SAA. SAA reacted with the HEK293-derived clones that stably express human ABCA1 (293/2c) or ABCA7 (293/6c) to generate cholesterol-containing HDL in a similar manner to apoA-I. Dibutyryl cyclic AMP and phorbol 12-myristate 13-acetate, which differentiate apoA-I-mediated cellular lipid release between 293/2c and 293/6c, also exhibited the same differential effects on the SAA-mediated reactions. No evidence was found for the ABCA1/ABCA7-independent lipid release by SAA. Characterization of physicochemical properties of the HDL revealed that SAA-generated HDL particles had higher density, larger diameter, and slower electrophoretic mobility than those generated by apoA-I. These results demonstrate that SAA generates cholesterol-containing HDL directly with cellular lipid and that the reaction is mediated by ABCA1 and ABCA7.     

ATF6调节ABCA1基因的表达

ATP结合盒式运载蛋白A1(ATP-binding cassette transporter A1,ABCA1)是近年来发现的极其重要的脂质转运大分子膜蛋白,它可将过量胆固醇从细胞内向细胞外输送到载脂蛋白并包装成高密度脂蛋白(HDL)的膜蛋白,促进胆固醇的逆转运.初步研究转录因子ATF6对ABCA1的表达调控,结果发现,ATF6在人胚胎肾细胞HEK293内剂量依赖性地调节ABCA1基因转录及蛋白质表达. ATF6调节ABCA1与内质网应激信号通路无关. 启动子序列缺失与突变分析表明ATF6作用区位于ABCA1启动子上游-156～-928bp之间, 可能需要E-box的参与,但不需要DR4元件.进而,动物试验结果显示用腺病毒在C57小鼠肝脏过表达ATF6,在mRNA水平上调ABCA1. 本文的研究发现了ATF6新的功能以及调控ABCA1的新机制.     

Role of blood-brain barrier organic anion transporter 3 (OAT3) in the efflux of indoxyl sulfate,a uremic toxin: its involvement in neurotransmitter metabolite clearance from the brain

Renal impairment is associated with CNS dysfunctions and the accumulation of uremic toxins, such as indoxyl sulfate, in blood. To evaluate the relevance of indoxyl sulfate to CNS dysfunctions, we investigated the brain-to-blood transport of indoxyl sulfate at the blood-brain barrier (BBB) using the Brain Efflux Index method. [(3)H]Indoxyl sulfate undergoes efflux transport with an efflux transport rate of 1.08 x 10(-2)/min, and the process is saturable with a Km of 298 microm. This process is inhibited by para-aminohippuric acid, probenecid, benzylpenicillin, cimetidine and uremic toxinins, such as hippuric acid and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid. RT-PCR revealed that an OAT3 mRNA is expressed in conditionally immortalized rat brain capillary endothelial cell lines and rat brain capillary fraction. Xenopus oocytes expressing OAT3 were found to exhibit [(3)H]indoxyl sulfate uptake, which was significantly inhibited by neurotransmitter metabolites, such as homovanillic acid and 3-methoxy-4-hydroxymandelic acid, and by acyclovir, cefazolin, baclofen, 6-mercaptopurine, benzoic acid, and ketoprofen. These results suggest that OAT3 mediates the brain-to-blood transport of indoxyl sulfate, and is also involved in the efflux transport of neurotransmitter metabolites and drugs. Therefore, inhibition of the brain-to-blood transport involving OAT3 would occur in uremia and lead to the accumulation of neurotransmitter metabolites and drugs in the brain.     

Influence of breast cancer resistance protein (Abcg2) and p-glycoprotein (Abcb1a) on the transport of imatinib mesylate (Gleevec) across the mouse blood-brain barrier

Imatinib, a protein tyrosine kinase inhibitor, may prevent the growth of glioblastoma cells. Unfortunately, its brain distribution is restricted by p-glycoprotein (p-gp or multidrug resistance protein Mdr1a), and probably by breast cancer resistance protein (Bcrp1), two efflux pumps expressed at the blood-brain barrier (BBB). We have used in situ brain perfusion to investigate the mechanisms of imatinib transport across the mouse BBB. The brain uptake of imatinib in wild-type mice was limited by saturable efflux processes. The inhibition of p-gp, by valspodar and zosuquidar, increased imatinib uptake (2.5-fold), as did the deficiency of p-gp in Mdr1a/1b(-/-) mice (5.5-fold). Perfusing imatinib with the p-gp/Bcrp1 inhibitor, elacridar, enhanced the brain uptake of imatinib in wild-type (4.1-fold) and Mdr1a/1b(-/-) mice (1.2-fold). However, the brain uptake of imatinib was similar in wild-type and Bcrp1(-/-) mice when it was perfused at a non-saturating concentration. The brain uptake of CGP74588, an active metabolite of imatinib, was low. It was increased by perfusion with elacridar (twofold), but not with valspodar and zosuquidar. CGP74588 uptake was 1.5 times greater in Bcrp1(-/-) mice than in wild-type mice. These data suggest that imatinib transport at the mouse BBB is limited by p-gp and probably by Bcrp1, and that CGP74588 transport is restricted by Bcrp1.     

The central helices of ApoA-I can promote ATP-binding cassette transporter A1 (ABCA1)-mediated lipid efflux. Amino acid residues 220-231 of the wild-type ApoA-I are required for lipid efflux in vitro and high density lipoprotein formation in vivo

We have mapped the domains of lipid-free apoA-I that promote cAMP-dependent and cAMP-independent cholesterol and phospholipid efflux. The cAMP-dependent lipid efflux in J774 mouse macrophages was decreased by approximately 80-92% by apoA-I[delta(185-243)], only by 15% by apoA-I[delta(1-41)] or apoA-I[delta(1-59)], and was restored to 75-80% of the wild-type apoA-I control value by double deletion mutants apoA-I[delta(1-41)delta(185-243)] and apoA-I[delta(1-59)delta(185-243)]. Similar results were obtained in HEK293 cells transfected with an ATP-binding cassette transporter A1 (ABCA1) expression plasmid. The double deletion mutant of apoA-I had reduced thermal and chemical stability compared with wild-type apoA-I. Sequential carboxyl-terminal deletions showed that cAMP-dependent cholesterol efflux was diminished in all the mutants tested, except the apoA-I[delta(232-243)] which had normal cholesterol efflux. In cAMP-untreated or in mock-transfected cells, cholesterol efflux was not affected by the amino-terminal deletions, but decreased by 30-40% and 50-65% by the carboxyl-terminal and double deletions, respectively. After adenovirus-mediated gene transfer in apoA-I-deficient mice, wild-type apoA-I and apoA-I[delta(1-41)] formed spherical high density lipoprotein (HDL) particles, whereas apoA-I[delta(1-41)delta(185-243)] formed discoidal HDL. The findings suggest that although the central helices of apoA-I alone can promote ABCA1-mediated lipid efflux, residues 220-231 are necessary to allow functional interactions between the full-length apoA-I and ABCA1 that are required for lipid efflux and HDL biogenesis.     

ATP-binding cassette transporter A7 (ABCA7) binds apolipoprotein A-I and mediates cellular phospholipid but not cholesterol efflux

ATP-binding cassette transporter 1 (ABCA1), the defective transporter in Tangier disease, binds and promotes cellular cholesterol and phospholipid efflux to apolipoprotein I (apoA-I). Based on a high degree of sequence homology between ABCA1 and ABCA7, a transporter of unknown function, we investigated the possibility that ABCA7 might be involved in apolipoprotein binding and lipid efflux. Similarly to cells expressing ABCA1, HEK293 cells overexpressing ABCA7 showed specific binding and cross-linking of lipid-poor apoA-I. ABCA7 expression increased cellular phosphatidylcholine and sphingomyelin efflux to apoA-I in a manner similar to ABCA1 but had no effect on cholesterol efflux. Western analysis showed a high protein level of ABCA7 in mouse spleen, lung, adrenal, and brain but low expression in liver. In contrast to ABCA1, ABCA7 showed moderate basal mRNA and protein levels in macrophages and lymphocytes but no induction by liver X receptor activation. These studies show that ABCA7 has the ability to bind apolipoproteins and promote efflux of cellular phospholipids without cholesterol, and they suggest a possible role of ABCA7 in cellular phospholipid metabolism in peripheral tissues.     

Apolipoprotein A-I activates Cdc42 signaling through the ABCA1 transporter

It has been suggested that the signal transduction initiated by apolipoprotein A-I (apoA-I) activates key proteins involved in cholesterol efflux. ABCA1 serves as a binding partner for apoA-I, but its participation in apoA-I-induced signaling remains uncertain. We show that the exposure of human fibroblasts to ABCA1 ligands (apolipoproteins and amphipathic helical peptides) results in the generation of intracellular signals, including activation of the small G-protein Cdc42, protein kinases (PAK-1 and p54JNK), and actin polymerization. ApoA-I-induced signaling was abrogated by glyburide, an inhibitor of the ABC transporter family, and in fibroblasts from patients with Tangier disease, which do not express ABCA1. Conversely, induction of ABCA1 expression with the liver X receptor agonist, T0901317, and the retinoid X receptor agonist, R0264456, potentiated apoA-I-induced signaling. Similar effects were observed in HEK293 cells overexpressing ABCA1-green fluorescent protein (GFP) fusion protein, but not ABCA1-GFP (K939M), which fails to hydrolyze ATP, or a nonfunctional ABCA1-GFP with a truncated C terminus. We further found that Cdc42 coimmunoprecipitates with ABCA1 in ABCA1-GFP-expressing HEK293 cells exposed to apoA-I but not in cells expressing ABCA1 mutants. We conclude that ABCA1 transduces signals from apoA-I by complexing and activating Cdc42 and downstream kinases and, therefore, acts as a full apoA-I receptor.     

Organic anion transporter 3 is involved in the brain-to-blood efflux transport of thiopurine nucleobase analogs

Thiopurines are used as antileukemic drugs. However, during chemotherapy CNS relapses occur due to the proliferation of leukemic cells in the CNS resulting from restricted drug distribution in the brain. The molecular mechanism for this limited cerebral distribution remains unclear. The purpose of this study was to identify the transporter responsible for the brain-to-blood transport of thiopurines across the blood-brain barrier (BBB) using the brain efflux index method. [14C]6-Mercaptopurine (6-MP) and [3H]6-thioguanine were eliminated from rat brain in a time-dependent manner. The elimination of [14C]6-MP was inhibited by substrates of rat organic anion transporters (rOATs), including indomethacin and benzylpenicillin. rOAT1 and rOAT3 exhibited 6-MP uptake, while benzylpenicillin inhibited rOAT3-mediated uptake, but not that by rOAT1. rOAT3-mediated [14C]6-MP uptake was also inhibited by other thiopurine derivatives. Although methotrexate inhibited rOAT3-mediated [14C]6-MP uptake, the Ki value was 17.5-fold greater than the estimated brain concentration of methotrexate in patients receiving chemotherapy. Accordingly, 6-MP would undergo efflux transport by OAT3 from the brain without any inhibitory effect from coadministered methotrexate in the chemotherapy. In conclusion, rOAT3 is involved in the brain-to-blood transport of thiopurines at the BBB and is one mechanism of limited cerebral distribution.     

Implications of cerebrovascular ATP-binding cassette transporter G1 (ABCG1) and apolipoprotein M in cholesterol transport at the blood-brain barrier

Impaired cholesterol/lipoprotein metabolism is linked to neurodegenerative diseases such as Alzheimer's disease (AD). Cerebral cholesterol homeostasis is maintained by the highly efficient blood-brain barrier (BBB) and flux of the oxysterols 24(S)-hydroxycholesterol and 27-hydroxycholesterol, potent liver-X-receptor (LXR) activators. HDL and their apolipoproteins are crucial for cerebral lipid transfer, and loss of ATP binding cassette transporters (ABC)G1 and G4 results in toxic accumulation of oxysterols in the brain. The HDL-associated apolipoprotein (apo)M is positively correlated with pre-β HDL formation in plasma; its presence and function in the brain was thus far unknown. Using an in vitro model of the BBB, we examined expression, regulation, and functions of ABCG1, ABCG4, and apoM in primary porcine brain capillary endothelial cells (pBCEC). RT Q-PCR analyses and immunoblotting revealed that in addition to ABCA1 and scavenger receptor, class B, type I (SR-BI), pBCEC express high levels of ABCG1, which was up-regulated by LXR activation. Immunofluorescent staining, site-specific biotinylation and immunoprecipitation revealed that ABCG1 is localized both to early and late endosomes and on apical and basolateral plasma membranes. Using siRNA interference to silence ABCG1 (by 50%) reduced HDL-mediated [³H]-cholesterol efflux (by 50%) but did not reduce [³H]-24(S)-hydroxycholesterol efflux. In addition to apoA-I, pBCEC express and secrete apoM mainly to the basolateral (brain) compartment. HDL enhanced expression and secretion of apoM by pBCEC, apoM-enriched HDL promoted cellular cholesterol efflux more efficiently than apoM-free HDL, while apoM-silencing diminished cellular cholesterol release. We suggest that ABCG1 and apoM are centrally involved in regulation of cholesterol metabolism/turnover at the BBB.     

The low density lipoprotein receptor-related protein 1 mediates uptake of amyloid beta peptides in an in vitro model of the blood-brain barrier cells

The metabolism of amyloid beta peptide (A beta) in the brain is crucial to the pathogenesis of Alzheimer disease. A body of evidence suggests that A beta is actively transported from brain parenchyma to blood across the blood-brain barrier (BBB), although the precise mechanism remains unclear. To unravel the cellular and molecular mechanism of A beta transport across the BBB, we established a new in vitro model of the initial internalization step of A beta transport using TR-BBB cells, a conditionally immortalized endothelial cell line from rat brain. We show that TR-BBB cells rapidly internalize A beta through a receptor-mediated mechanism. We also provide evidence that A beta internalization is mediated by LRP1 (low density lipoprotein receptor-related protein 1), since administration of LRP1 antagonist, receptor-associated protein, neutralizing antibody, or small interference RNAs all reduced A beta uptake. Despite the requirement of LRP1-dependent internalization, A beta does not directly bind to LRP1 in an in vitro binding assay. Unlike TR-BBB cells, mouse embryonic fibroblasts endogenously expressing functional LRP1 and exhibiting the authentic LRP1-mediated endocytosis (e.g. of tissue plasminogen activator) did not show rapid A beta uptake. Based on these data, we propose that the rapid LRP1-dependent internalization of A beta occurs under the BBB-specific cellular context and that TR-BBB is a useful tool for analyzing the molecular mechanism of the rapid transport of A beta across BBB.     

Cross-inhibition of SR-BI- and ABCA1-mediated cholesterol transport by the small molecules BLT-4 and glyburide

Scavenger receptor class B type I (SR-BI) and ABCA1 are structurally dissimilar cell surface proteins that play key roles in HDL metabolism. SR-BI is a receptor that binds HDL with high affinity and mediates both the selective lipid uptake of cholesteryl esters from lipid-rich HDL to cells and the efflux of unesterified cholesterol from cells to HDL. ABCA1 mediates the efflux of unesterified cholesterol and phospholipids from cells to lipid-poor apolipoprotein A-I (apoA-I). The activities of ABCA1 and other ATP binding cassette superfamily members are inhibited by the drug glyburide, and SR-BI-mediated lipid transport is blocked by small molecule inhibitors called BLTs. Here, we show that one BLT, [1-(2-methoxy-phenyl)-3-naphthalen-2-yl-urea] (BLT-4), blocked ABCA1-mediated cholesterol efflux to lipid-poor apoA-I at a potency similar to that for its inhibition of SR-BI (IC(50) approximately 55-60 microM). Reciprocally, glyburide blocked SR-BI-mediated selective lipid uptake and efflux at a potency similar to that for its inhibition of ABCA1 (IC(50) approximately 275-300 microM). As is the case with BLTs, glyburide increased the apparent affinity of HDL binding to SR-BI. The reciprocal inhibition of SR-BI and ABCA1 by BLT-4 and glyburide raises the possibility that these proteins may share similar or common steps in their mechanisms of lipid transport.     

Selective gene silencing of rat ATP-binding cassette G2 transporter in an in vitro blood-brain barrier model by short interfering RNA

The aim of the present study was to specifically silence the rat ATP-binding cassette transporter G2 (rABCG2) gene in brain capillary endothelial cells by transfection of short interfering RNA (siRNA). Four different siRNAs designed to target rABCG2 were each transfected into HEK293 cells with myc-tagged rABCG2 cDNA. Quantitative real-time PCR and western blot analyses revealed that three of the siRNAs were able to reduce exogenous rABCG2 mRNA and protein levels in HEK293 cells. Moreover, rABCG2-mediated mitoxantrone efflux transport was suppressed by the introduction of these three siRNAs into HEK293 cells. In contrast, the other siRNA and non-specific control siRNA did not significantly affect the mRNA expression, the protein level or the transport activity. Endogenous rABCG2 mRNA and protein expression in a conditionally immortalized rat brain capillary endothelial cell line (TR-BBB13) was suppressed by the most potent siRNA among the four siRNAs tested. Furthermore, this siRNA did not affect the mRNA levels of other ABC transporters, such as ABCB1, ABCC1 and ABCG1, and the protein level of ABCB1 in TR-BBB13 cells, suggesting that it can selectively silence rABCG2 at the blood-brain barrier. This should be a useful and novel strategy for clarifying the contribution of rABCG2 to brain-to-blood transport of substrate drugs and endogenous compounds across the blood-brain barrier.     

Phosphorylation of a pest sequence in ABCA1 promotes calpain degradation and is reversed by ApoA-I

ATP-binding cassette transporter A1 (ABCA1), the defective molecule in Tangier disease, mediates the apoAI-dependent efflux of excess cholesterol from cells. We recently showed that ABCA1 proteolysis by calpain was dependent on a PEST sequence in the cytoplasmic region of ABCA1 and was reversed by apoA-I interaction with ABCA1. We show here that phosphorylation of ABCA1 in HEK293 cells was reduced by 63 +/- 2.4% after removal of the PEST sequence (ABCA1delPEST) or by incubation of cells with apoAI (58 +/- 3.3%). By contrast, ABCA1delPEST showed no further decrease of phosphorylation upon apoAI treatment. To assess the hypothesis that PEST sequence phosphorylation could regulate ABCA1 calpain proteolysis, we mutagenized S/T residues in the PEST sequence and identified Thr-1286 and Thr-1305 as constitutively phosphorylated residues. The ABCA1-T1286A/T1305A mutant was not degraded by calpain and was not further stabilized upon apoA-I treatment. The T1286A/T1305A mutant showed a 3.1-fold increase in cell surface expression and a 2.3-fold increase of apoAI-mediated cholesterol efflux compared with wild type ABCA1. In conclusion, we propose a mechanism of regulation of ABCA1 cell surface expression and function in which the interaction with apoA-I results in dephosphorylation of the ABCA1 PEST sequence and thereby inhibits calpain degradation leading to an increase of ABCA1 cell surface expression.     

Development of a selective photoactivatable antagonist for corticotropin-releasing factor receptor, type 2 (CRF2).

A novel photoactivatable analog of antisauvagine-30 (aSvg-30), a specific antagonist for corticotropin-releasing factor (CRF) receptor, type 2 (CRF2), has been synthesized and characterized. The N-terminal amino-acid d-Phe in aSvg-30 [d-Phe11,His12]Svg(11-40) was replaced by a phenyldiazirine, the 4-(1-azi-2,2,2-trifluoroethyl)benzoyl (ATB) residue. The photoactivatable aSvg-30 analog ATB-[His12]Svg was tested for its ability to displace [125I-Tyr0]oCRF or [125I-Tyr0]Svg from membrane homogenates of human embryonic kidney (HEK) 293 cells stably transfected with cDNA coding for rat CRF receptor, type 1 (rCRF1) or mouse CRF receptor, type 2beta (mCRF2beta). Furthermore, the ability of ATB-[His12]Svg(12-40) to inhibit oCRF- or Svg-stimulated cAMP production of transfected HEK 293 cells expressing either rCRF1 (HEK-rCRF1 cells) or mCRF2beta (HEK-mCRF2beta cells) was determined. Unlike astressin and photo astressin, ATB-[His12]Svg(12-40) showed high selective binding to mCRF2beta (Ki = 3.1 +/- 0.2 nm) but not the rCRF1 receptor (Ki = 142.5 +/- 22.3 nm) and decreased Svg-stimulated cAMP activity in mCRF2beta-expressing cells in a similar fashion as aSvg-30. A 66-kDa protein was identified by SDS/PAGE, when the radioactively iodinated analog of ATB-[His12]Svg(12-40) was covalently linked to mCRF2beta receptor. The specificity of the photoactivatable 125I-labeled CRF2beta antagonist was demonstrated with SDS/PAGE by the finding that this analog could be displaced from the receptor by antisauvagine-30, but not other unrelated peptides such as vasoactive intestinal peptide (VIP).