Identification and characterization of receptors specific for human pancreatic secretory trypsin inhibitor

Abstract

Specific binding sites for human pancreatic secretory trypsin inhibitor (PSTI) on 3T3 Swiss albino cells were studied using radioiodinated recombinant PSTI. Some ion species, pH, and temperature significantly influenced the binding of 125I-PSTI. Kinetic studies showed that the binding of 125I-PSTI to 3T3 Swiss albino cells reached the maximum level within 120 min at 4°C, with a slow dissociation rate. The half-maximal inhibition (ID50) of 125I-PSTI binding by unlabeled PSTI occurred at 1.0 × 10-10 M. On Scatchard analysis of the competitive binding data, linear plots indicated a single class of receptors with high affinity (Kd = 5.3 × 10-10 M) on 3T3 Swiss albino cells, the number of receptors being 5,400 per cell. Treatment of surface-bound radiolabeled PSTI with a chemical crosslinker (disuccinimidyl suberate) led to the identification of a membrane polypeptide of Mr 140,000 to which PSTI was crosslinked. The formation was inhibited by an excess amount of unlabeled PSTI in a dose-dependent manner. The binding of 125I-PSTI to 3T3 Swiss albino cells was competitively inhibited by unlabeled PSTI but not by other peptide hormones, such as epidermal growth factor (EGF), bovine fibroblast growth factor, insulin-like growth factor, transforming growth factor a, platelet-derived growth factor, and tumor necrosis factor, indicating the presence of receptors specific for PSTI. Various protease inhibitors had no or only a little effect, and mercaptoethanol and dithiothreitol strongly decreased the binding of 125I-PSTI. Incubation at 37°C resulted in rapid internalization of cell-bound 125I-PSTI, followed by the appearance of trichloroacetic acid-soluble 125I-radioactivity in the culture medium, due to degradation of internalized PSTI. In addition, PSTI stimulated [3H]thymidine incorporation into DNA on 3T3 Swiss albino cells in a dose-dependent manner. The combined addition of PSTI and EGF stimulated [3H]thymidine incorporation to an extent greater than that seen with either agent alone. These results indicated that the biological effect of PSTI was mediated by high affinity plasma membrane receptors, which were not a cell-surface proteinase(s). Specific binding of 125I-PSTI was noted with the following cells: WI-38, 3T3 Swiss albino, HUVE, BDC-1, and H4-II-E-C3.