Localization of transforming growth factor beta binding site in betaglycan. Comparison with small extracellular matrix proteoglycans.
The most abundant binding molecule for transforming growth factor beta (TGF-beta) on many cell types is betaglycan, a transmembrane proteoglycan. To localize the binding site for TGF-beta in betaglycan, parts of the betaglycan extracellular portion were expressed as recombinant fusion proteins in bacteria and tested for their ability to compete for the binding of TGF-beta 1 to Hep G2 cells. One fragment encompassing 226 residues near the transmembrane domain (amino acids 543-769) (López-Casillas, F., Cheifetz, S., Doody, J., Andres, J. L., Lane, W. S., and Massagué, J. (1991) Cell 67, 785-795) was active, whereas fusion proteins representing the other parts of the betaglycan ectodomain were inactive. Affinity measurements revealed two classes of binding sites (Kd = 3.9 nM and Kd = 145 nM) for the active fusion protein. The binding of the betaglycan fusion protein to immobilized TGF-beta 1 was inhibited by fusion proteins representing the core proteins of the small interstitial proteoglycans decorin, biglycan and fibromodulin, each also known to bind TGF-beta. The effective concentrations of TGF-beta for binding to these other proteoglycans were similar to those required for binding to betaglycan, indicating similar affinities for the binding of proteoglycans and betaglycan. Affinity cross-linking showed that, at low concentrations, the betaglycan fragment enhanced the binding of TGF-beta to the type II receptor and to endogenous betaglycan but had no effect on the binding to type I receptor. At high concentrations, the TGF-beta binding fragment inhibited the binding of TGF-beta to all these receptors. The fragment enhanced the activity of TGF-beta in mink lung cell bioassay at all active concentrations. The results indicate that betaglycan and the decorin type proteoglycans all bind to the same or closely spaced sites in TGF-beta and compete with one another for the binding. In addition, betaglycan may cooperate with the type II receptor on TGF-beta binding.
Fukushima
D
D
Cancer Research Center, La Jolla Cancer Research Foundation, California 92037.
Bützow
R
R
Hildebrand
A
A
Ruoslahti
E
E
D000818:Animals; D001665:Binding Sites; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005109:Extracellular Matrix / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000096:biosynthesis / Q000378:metabolism* / Q000494:pharmacology; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D011509:Proteoglycans / Q000096:biosynthesis / Q000378:metabolism* / Q000494:pharmacology; D051381:Rats; D018125:Receptors, Transforming Growth Factor beta; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D015183:Restriction Mapping; D016212:Transforming Growth Factor beta / Q000378:metabolism* / Q000494:pharmacology
D018076:DNA, Complementary; D008565:Membrane Proteins; D010446:Peptide Fragments; D011509:Proteoglycans; D018125:Receptors, Transforming Growth Factor beta; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; C071736:betaglycan