Abstract 996: Inhibition of Endothelial Cell Migration by Semaphorin 3C is Modulated by the Synectin-Dependent Uptake of the Semaphorin Receptor Neuropilin-1
Introduction: Neuropilin is one of the major endothelial cell-surface receptors involved in the transmission of both attractive and repulsive guidance cues. These cues are delivered either by vascular endothelial growth factor (VEGF) or by members of the semaphorin family of guidance cues, respectively. The only known binding partner of the cytoplasmic domain of neuropilin-1 (Npn-1) is synectin, a ubiquitous adaptor protein that contains a PDZ modular binding domain. Synectin facilitates inward movement of endocytic vesicles by serving as a docking site for the actin-dependent retrograde molecular motor myosin VI.
Hypothesis: The signaling activity of numerous cell-surface receptors is modulated by their uptake and persists after their internalization. Does synectin-mediated uptake of Npn-1 regulate cellular response to semaphorin?
Methods and results: Using pulse-chase uptake assays with heart endothelial cells (EC) from wild type mice, we found that Npn-1 is internalized upon binding semaphorin 3C, an Npn-1 ligand and a known morphogen of the cardiovascular system. FACS-measured Npn-1 uptake occurred in a synectin-dependent manner, as it was significantly slower in heart EC from synectin-null mice. Moreover, the uptake of semaphorin 3C itself was similarly delayed in synectin-null EC. Npn-1 uptake was triggered by sema3C rather than being constitutive. On the other hand, the uptake rate of sema3E, another known morphogen of the cardiovascular system and the single member of the secreted semaphorin subfamily which does not bind neuropilin, was similar in wild type and in synectin-null endothelial cells. In order to examine the potential functional effects of the delayed Npn-1 and semaphorin 3C uptakes in synectin-null EC, we compared the sema3C inhibition of their migration rate to the sema3C inhibition of the migration rate of wild type EC. We found that the migration rate of synectin-null EC was not reduced by semaphorin 3C treatment (98±10% relative to the migration rate of the control group) while the migration rate of wild type EC was reduced to 49±2% relative to the control group.
Conclusions: These findings suggest that the inhibition of EC migration by semaphorin 3C requires Npn-1 uptake, and implicate synectin in Npn-1 signaling.