Identification of the high affinity binding site in transforming growth factor-β involved in complex formation with α2-macroglobulin -: Implications regarding the molecular mechanisms of complex formation between α2-macroglobulin and growth factors, cytokines, and hormones

The biological activities of transforming growth factor-beta isoforms (TGF-beta (1,2)) are known to be modulated by alpha (2)-macroglobulin (alpha M-2). alpha M-2 forms complexes with numerous growth factors, cytokines, and hormones, including TGF-beta. Identification of the binding sites in TGF-beta isoforms responsible for high affinity interaction with a2M many unravel the molecular basis of the complex formation. Here we demonstrate that among nine synthetic pentacosapeptides with overlapping amino acid sequences spanning the entire TGF-beta (1), molecule, the peptide (residues 41-65) containing Trp-52 exhibited the most potent activity in inhibiting the formation of complexes between I-125-TGF-beta (1) and activated alpha M-2 (alpha M-2*) as determined by nondenaturing polyacrylamide gel electrophoresis and by plasma clearance in mice. TGF-beta (2) peptide containing the homologous sequence and Trp-52 was as active as the TGF-beta (1) peptide, whereas the corresponding TGF-beta (3) peptide lacking Trp-52, was inactive. The replacement of the Trp-52 with alanine abolished the inhibitory activities of these peptides. I-125-TGF-beta (3), which lacks Trp-52, bound to a2M* with an affinity lower than that of I-125-TGF-beta (1). Furthermore, unlabeled TGF-beta (3) and the mutant TGF-beta (1)W52A, in which Trp-52 was replaced with alanine, were less potent than unlabeled TGF-P, in blocking I-125-TGF-beta (1) binding to alpha M-2*.TGF-beta (1) and TGF-beta (2) peptides containing Trp-52 were also effective in inhibiting I-125-nerve growth factor binding to a2M*. These results suggest that Trp-52 is involved in high affinity binding of TGF-beta to alpha M-2*. They also imply that TGF-beta and other growth factors/cytokines/hormones may form complexes with a2M* via a common mechanism involving the interactions between topologically exposed Trp and/or other hydrophobic residues and a hydrophobic region in alpha M-2*.