A potential mechanism for the trafficking of tissue plasminogen activator (t-pa) to the regulated pathway of secretion: binding to the major secretory vesicle protein, chromogranin a (cga)

t-PA has a widespread neuroendocrine distribution including its expression in the chromaffin cells of the sympathoadrenal system. Chromaffin cell t-PA is sorted into the regulated pathway of secretion [into catecholamine storage vesicles (CSV)] and is (released with catecholamines in response to sympathoadrenal activation, suggesting that CSV may serve as a reservoir for the rapid release of t-PA. CgA, the major secretory protein in CSV and in secretory vesicles throughout the neuroendocrine system, may play a crucial role in the targeting of proteins into the regulated secretory pathway, by forming aggregated "granin" complexes to which other proteins destined for the regulated secretory vesicle bind and are separated from constitutively secreted proteins in the trms-Golgi network (TON) with subsequent movement into the dense core regulated secretory vesicle. Formation of such complexes is facilitated by conditions of the TON (low pH, high Ca2). To investigate potential mechanisms for the targeting of t-PA to CSV, we tested the hypothesis that spécifie binding of t-PA to CgA occurs and is enhanced under conditions of the TGN. t-PA was immobilized onto microtiter wells and incubated with 1 nM human ml-CgA in the presence or absence of competitors. IBI-CgA bound specifically to t-PA at pH 7.4 with EOTA. The binding was g5l% inhibited by luM unlabeled CgA. Unrelated proteins (ovalbumin, transform, RNase) inhibited the binding by 20%, demonstrating the specificity of the interaction. In addition, mI-CgA binding was 721% inhibited by lysine analogs, suggesting that the interaction may be mediated by the kringle 2 domain of t-PA. When binding was analyzed under conditions within the TON (pH 6.4, 10 mM Ca) or within the secretory vesicle (pH 5.5, 20 mM Ca?) specific binding of '"I-CgA to t-PA was enhanced 4.2-fold and 4.6-fold, respectively. Real time t-PA/CgA binding parameters using surface plasmon resonance revealed an association rate constant of 2.49 X 103 M'V1 and a dissociation rate constant of 5.16 X 10~! s"', and an estimated Kd of 21 nM, indicative of a high affinity interaction. These studies suggest that t-PA interacts specifically with CgA in a pH- and Ca2- dependent fashion with enhancement of binding under conditions encountered in the TON. Binding of t-PA to aggregated CgA complexes may provide a mechanism for the targeting of t-PA to the regulated pathway of secretion in chrornaffin cells as well as in other neuroendocrine tissues.