Ectodomain cleavage and shedding of the type III transforming growth factor-beta receptor in lung membranes effect of temperature, ligand binding and membrane solubilization.
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 [125I]-transforming growth factor-beta1 (TGF-beta1) on a per gram basis. This observation, together with the lack of information on TGF-beta receptor expression in the lung, prompted us to attempt to characterize TGF-beta receptors in this tissue. In the present report we show that the type III TGF-beta receptor is the most abundant TGF-beta binding protein in rat lung membranes and that it exhibits a 10-fold higher affinity for TGF-beta2 than for TGF-beta1. 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-beta receptor ectodomain.
Philip
A
A
Receptor Group, Biotechnology Research Institute, National Research Council Canada, Montréal Canada.
Hannah
R
R
O'connor-McCourt
M
M
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002462:Cell Membrane / Q000378:metabolism; D008024:Ligands; D008168:Lung / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011485:Protein Binding; D011509:Proteoglycans / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D051381:Rats; D011917:Rats, Inbred Lew; D017207:Rats, Sprague-Dawley; D018125:Receptors, Transforming Growth Factor beta / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D013696:Temperature; D016212:Transforming Growth Factor beta / Q000378:metabolism
D000345:Affinity Labels; D000906:Antibodies; D008024:Ligands; D011509:Proteoglycans; D018125:Receptors, Transforming Growth Factor beta; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; C071736:betaglycan