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The DNA-binding properties of the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were investigated using chromatography on DNA-cellulose columns. A maximal binding of about 40% of the total receptor complex to DNA-cellulose was observed. In order to interact with DNA, the receptor must first bind TCDD. A heat-activation step followed by gel permeation chromatography using Sephadex G-25 increased the binding of the cytosolic receptor to DNA. The DNA-binding ability of the receptor was almost lost following mild proteolysis using trypsin or a-chymotrypsin, although these treatments did not reduce its ligand binding capacity and had no apparent effect on its size. Furthermore, pre-treatment of the DNA-cellulose column with an intercalating drug, ethidium bromide, resulted in inhibition of the binding of the TCDD-receptor complex to DNA, indicating that not only electrostatic interactions but also the configuration of DNA are of importance in receptor-DNA interactions.In analogy to the endocrine model of gene regulation by steroid hormones, several lines of evidence indicate that intracellular, soluble receptor proteins are essential mediators of the biological responses produced by certain xenobiotics, i. e. halogenated or non-halogenated aromatic hydrocarbons One particularly striking and often-studied biochemical effect produced by TCDD and related compounds is their potency to induce specific isozymes of cytochrome P-450 and the associated monooxygenase activity aryl hydrocarbon hydroxylase [l , 41 together with several non-monooxygenase activities, e. g. UDP-glucuronyltransferase, NAD(P)H dehydrogenase (quinone) (formerly called DT-diaphorase) and ornithine decarboxylase [l]. Several studies have demonstrated that administration of TCDD or TCDD-like inducers leads to a large increase in translatable mRNAs encoding the TCDDinduced isozyme(s) of cytochrome P-450 [5 -71. By in vitro nuclear transcription studies, this process has been shown to reflect an increased rate of transcription of the specific cytochrome P-450 gene(s) [8-lo]. In analogy to the 'twostep' model proposed for the mechanism of action of steroid Correspondence to J.-A. Gustafsson, Institutionen for Medicinsk Naringslara, Huddinge Sjukhus, F69, S-141 86 Huddinge, SwedenAbbreviations. TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDBF, 2,3,7,8-tetrachlorodibenzofuran; Azso -310 unit, the amount of material giving an absorbance at 280nm of 1 more than the absorbance at 310 nm in a 1 -cm pathlength cell; DCC, dextran-coated charcoal.Enzymes (IUB Recommendations 1984). Aryl hydrocarbon hydroxylase (EC 1.14.14.1); a-chymotrypsin (EC hormones [ l l , 121, an accumulation of TCDD-receptor complexes in the cell nucleus following occupation of cytosolic receptor sites has been reported by several authors [13 -171. This classical 'two-step' model has been questioned, however, and some recent reports [18, 191 suggest that unoccupied steroid hormone receptors as well as unoccupied TCDD receptors in the intact cell are contained in the nucleus, and ...

Previous studies from our laboratory [Philip, A. & O'Connor-McCourt, M. D. (1991) J. Biol. Chem. 266, 22290±22296] have shown that the lung exhibited the highest uptake of circulating [ 125 I]-transforming growth factor-b1 (TGF-b1) on a per gram basis. This observation, together with the lack of information on TGF-b receptor expression in the lung, prompted us to attempt to characterize TGF-b receptors in this tissue. In the present report we show that the type III TGF-b receptor is the most abundant TGF-b binding protein in rat lung membranes and that it exhibits a 10-fold higher affinity for TGF-b2 than for TGF-b1. We observed that the majority of the type III receptor population in lung membranes is cleaved at a site in the central portion of the ectodomain, the resulting two fragments (95 kDa and 58 kDa) being held together by disulfide bonds. Furthermore, we demonstrate that a soluble form of the ectodomain of the type III receptor is shed from rat lung membranes in an efficient manner, with protease cleavage occurring at a site close to the transmembrane domain. This shedding is controllable by temperature, thus providing a system to study the mechanism of ectodomain release. Using this system, we show that the shedding is inhibited by prior ligand binding and by membrane solubilization. The identification of a membrane preparation which exhibits controllable and quantitative release of the type III receptor ectodomain provides a unique cell-free system for further studies of the mechanism of shedding of the type III TGF-b receptor ectodomain.Keywords: type III transforming growth factor (TGF)-b receptor; ectodomain shedding; lung membrane.Transforming growth factor-bs (TGF-bs) are multifunctional peptides that regulate many critical cellular processes such as cell growth, differentiation, extracellular matrix deposition and immune function [1,2]. Three closely related TGF-b isoforms (TGF-b1, -b2, and -b3) are found in mammals. All three isoforms have been shown to be present in both fetal and adult lung in rodents and humans [3±5]. They have been demonstrated to regulate the synthesis of extracellular matrix molecules in the lung [6,7] and have been shown to be important in human and rodent lung epithelial cell proliferation and differentiation [4,8,9]. The regulated expression patterns of TGF-bs in the lung and their known biological activities suggest an important role for these factors during lung development and in mature lung homeostasis. In addition, they are strongly implicated in the pathogenesis of pulmonary fibrosis [10,11] [18]. The type I receptor is a 50±60-kDa protein that requires the presence of the type II receptor for TGF-b binding. The type II receptor, a 75±85-kDa protein, however, can bind the ligand independently, but requires the presence of the type I receptor for signaling [18]. The type III receptor, also known as betaglycan, is a membrane proteoglycan which is believed to enhance TGF-b binding to the signaling receptors [19,20]. The type III receptor has been reported to form ...