Vascular smooth muscle cell (vSMC) proliferation is important in atherosclerosis. We previously demonstrated that methylamine-activated alpha 2-macroglobulin (alpha 2M) and transforming growth factor beta 1 (TGF-beta 1) cause a synergistic proliferative response in quiescent rat aortic vSMCs [Stouffer, G. A., La-Marre, J., Gonias, S. L. & Owens, G. K. (1993) J. Biol. Chem. 268, 18,340-18,344]. The first goal of this study was to determine whether the synergy is due to the ability of alpha 2M-methylamine (alpha 2M-MeNH2) to bind TGF-beta 1 and target the growth factor to vSMCs that express the alpha 2M receptor. Receptor-recognized alpha 2M derivatives without TGF-beta 1-binding activity, including ternary alpha 2M-trypsin, an 18-kDa proteolytic fragment of the alpha 2M subunit, and the corresponding recombinant receptor-binding fragment (rRBF) increased vSMC [3H]thymidine incorporation and cell number in a manner similar to alpha 2M-MeNH2. In combination with TGF-beta 1, each alpha 2M derivative caused a synergistic vSMC proliferative response. vSMCs responded comparably when treated with alpha 2M-MeNH2 and TGF-beta 1 simultaneously or in sequence. Furthermore, alpha 2M-MeNH2-TGF-beta 1 complexes increased [3H]thymidine incorporation no more than alpha 2M-MeNH2 alone. These results indicate that TGF-beta 1 binding to alpha 2M is not responsible for the synergistic mitogenic activity. Additional studies were undertaken to determine whether activated alpha 2M independently induces a signal-transduction response in vSMCs. alpha 2M-MeNH2 and rRBF caused a rapid, transient increase in vSMC inositol 1,4,5-trisphosphate. This response was pertussis-toxin insensitive. Receptor-associated protein (RAP; 170 nmol/L) inhibited 91-95% of the specific binding of 125I-alpha 2M-MeNH2 and 125I-rRBF to vSMC; however, RAP did not affect the inositol 1,4,5-trisphosphate response or the mitogenic response. These studies suggest that vSMCs express a receptor, other than low-density-lipoprotein-receptor-related protein, that transduces a signal in response to activated alpha 2M. This receptor may mediate the mitogenic activity of alpha 2M in vSMC culture.