Affinity labeling of high-affinity α-thrombin binding sites on the surface of hamster fibroblasts

The serine proteinase α-thrombin potently stimulates reinitiation of DNA synthesis in quiescent Chinese hamster fibroblasts (CCL39 line). ¹²⁵I-labeled α-thrombin binds rapidly and specifically to CCL39 cells with high affinity (K_d ≈ 4 nM). Binding at 37°C was found to remain stable for 6 h or more during which time no receptor down-regulation, ligand internalization and/or degradation could be detected. The structure of α-thrombin receptors on CCL39 cells was identified by covalently coupling ¹²⁵I-α-thrombin to intact cells using a homobifunctional cross-linking agent, ethylene glycol bis(succinimidyl succinate). By resolution in sodium dodecyl sulfate polyacrylamide gel electrophoresis we observed the specific labeling of a major α-thrombin-binding site of M_r ≈ 150 000 revealed as a ¹²⁵I-α-thrombin cross-linked complex of M_r ≈ 180 000. Independent of chemical cross-linking, ¹²⁵I-α-thrombin also formed a covalent complex with a minor, 35 000 M_r, membrane component identified as protease nexin. Two derivatives of α-thrombin modified at the active site are 1000-fold less than α-thrombin for mitogenicity. These two non-mitogenic derivatives bound to cells with similar affinity and maximal binding capacity as native α-thrombin, and affinity-labeled the receptor subunit of M_r 150 000. When present in large excess, during incubation of cells with α-thrombin, these binding antagonists were ineffective in blocking α-thrombin-induced DNA synthesis. These data suggest that the specific 150 000 M_r binding sites that display high affinity for α-thrombin do not mediate induction of the cellular mitogenic response.