TRANSFORMING GROWTH-FACTOR-BETA ISOFORM 2-SPECIFIC HIGH-AFFINITY BINDING TO NATIVE ALPHA(2)-MACROGLOBULIN - CHIMERAS IDENTIFY A SEQUENCE THAT DETERMINES AFFINITY FOR NATIVE BUT NOT ACTIVATED ALPHA(2)-MACROGLOBULIN

Transforming growth factor beta 2 (TGF-beta 2) is less potent than TGF -beta 1 in some endothelial cell proliferation as says due to the grea ter tendency of TGF-beta 2 to bind alpha(2)-macroglobulin (alpha(2)M). Substitution of TGF-beta 1 residues 40-47 into the TGF-beta 2 sequenc e yields a chimeric molecule that, like TGF-beta 1, expresses activity that is not substantially affected by serum alpha(2)M (Burmester, J. K., Qian, S. W., Roberts, A. B., Huang, A., Amatayakul-Chantler, S., S uardet, L., Odartchenko, N., Madri, J. A., and Sporn, M. B. (1993) Pro c. Natl. Acad. Sci. U. S. A. 90, 8628-8632). In this investigation, we studied the binding of TGF-beta chimeras, which contain TGF-beta 1 re sidues 40-47, to both major conformations of human alpha(2)M under app arent equilibrium conditions. Native alpha(2)M, the primary form of th is protein in serum, bound TGF-beta(2)/beta(1)(40-82) and TGF-beta(2)/ beta(1)(40-47) with low affinity. The apparent K-D values for the two chimeras and native alpha(2)M were 310 and 330 nM, respectively. These values were much higher than the K-D determined for TGF-beta 2 and na tive alpha(2)M (11 nM) and equivalent to the K-D determined for TGF-be ta 1 and native alpha(2)M. By contrast, both TGF-beta chimeras bound a lpha(2)M-methylamine, an altered conformation of alpha(2)M, with high affinity (16 and 19 nM), which is characteristic of TGF-beta 2 and not TGF-beta 1. Fetal bovine heart endothelial cell DNA synthesis was inh ibited to a similar degree by TGF-beta 1, TGF-beta 2, TGF-beta(2)/beta (1)(40-82), and TGF-beta(2)/beta(1)(40-47) in the presence of dilute ( 0.2%) fetal bovine serum. When 0.07 mu M alpha 2M-methylamine was adde d, the activities of TGF-beta 2, TGF-beta(2)/beta(1)(40-82), and TGF-b eta(2)/beta(1)(40-47) were significantly counteracted while the activi ty of TGF-beta 1 was unchanged, as would be predicted by the equilibri um binding analyses. These studies indicate that the TGF-beta structur al elements, which mediate binding to native alpha(2)M and conformatio nally transformed alpha(2)M, are not equivalent. Residues 40-47 are cr itical in determining affinity for native alpha(2)M but are less impor tant in determining affinity for alpha(2)MM methylamine.