Identical or overlapping sequences in the primary structure of human alpha(2)-macroglobulin are responsible for the binding of nerve growth factor-beta, platelet-derived growth factor-BB, and transforming growth factor-beta.
alpha(2)-Macroglobulin (alpha(2)M) functions as a proteinase inhibitor and as a carrier of diverse growth factors. In this study, we localized binding sites for platelet-derived growth factor-BB (PDGF-BB) and nerve growth factor-beta (NGF-beta) to a linear sequence in the 180-kDa human alpha(2)M subunit which includes amino acids 591-774. A glutathione S-transferase fusion protein containing amino acids 591-774 (FP3) bound PDGF-BB and NGF-beta in ligand blotting assays whereas five other fusion proteins, which collectively include amino acids 99-590 and 775-1451 did not. The K(D) values for PDGF-BB and NGF-beta binding to immobilized FP3 were 300 +/- 40 and 180 +/- 30 nM, respectively; these values were comparable with those determined using methylamine-modified alpha(2)M, suggesting that higher-order alpha(2)M structure is not necessary for PDGF-BB and NGF-beta binding. PDGF-BB and NGF-beta blocked the binding of transforming growth factor-beta1 (TGF-beta1) to FP3. Furthermore, murinoglobulin, which is the only known member of the alpha-macroglobulin family that does not bind TGF-beta, also failed to bind PDGF-BB and NGF-beta. These results support the hypothesis that either a single linear sequence in human alpha(2)M or overlapping sequences are responsible for the binding of TGF-beta, PDGF-BB, and NGF-beta, even though there is minimal sequence identity between these three growth factors. FP3 blocked the binding of PDGF-BB to a purified chimeric protein, in which the extracellular domain of the PDGF beta receptor was fused to the IgG(1) Fc domain, and to PDGF receptors on NIH 3T3 cells. Thus, FP3 may inhibit the activity of PDGF-BB.
Gonias
S L
SL
Department of Pathology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA. slg2t@virginia.edu
Carmichael
A
A
Mettenburg
J M
JM
Roadcap
D W
DW
Irvin
W P
WP
Webb
D J
DJ
D016475:3T3 Cells; D000818:Animals; D000077214:Becaplermin; D001665:Binding Sites; D016228:Endothelial Growth Factors / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008222:Lymphokines / Q000378:metabolism; D051379:Mice; D020932:Nerve Growth Factor / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011485:Protein Binding; D020574:Proto-Oncogene Proteins c-sis; D017479:Receptors, Platelet-Derived Growth Factor / Q000037:antagonists & inhibitors / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012712:Serum Globulins / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D000511:alpha-Macroglobulins / Q000737:chemistry*
D016228:Endothelial Growth Factors; D008024:Ligands; D008222:Lymphokines; D010455:Peptides; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D011993:Recombinant Fusion Proteins; D012712:Serum Globulins; D016212:Transforming Growth Factor beta; D014409:Tumor Necrosis Factor-alpha; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D000511:alpha-Macroglobulins; C044177:murinoglobulin; D000077214:Becaplermin; D020932:Nerve Growth Factor; D005982:Glutathione Transferase; D017479:Receptors, Platelet-Derived Growth Factor