Abstract 10838: Tyrosine Phosphorylation of Syndecan-1, a Receptor for Remnant Lipoproteins, Triggers its Binding to Cortactin, an Essential Intracellular Mediator of a Novel Endocytic Pathway
BACKGROUND We previously reported that the syndecan-1 heparan sulfate proteoglycan directly mediates cellular uptake of model remnant lipoproteins through a novel endocytic pathway distinct from coated pits. Using a chimera, FcR-Synd1, that consists of an IgG Fc receptor ectodomain linked to the transmembrane and cytoplasmic regions of syndecan-1, we showed that efficient endocytosis is triggered by clustering upon binding multivalent ligands. Clustering causes movement into cholesterol-rich membrane rafts within 10 min. Uptake into the cell proceeds with a t1/2 of 1h and requires tyrosine kinases and the actin cytoskeleton. Replacement of a highly conserved juxtamembrane motif, MKKK, with four alanines was the only scanning mutation that we found to block tyrosine phosphorylation and efficient endocytosis of FcR-Synd1 after clustering.
GOAL In the current study, we sought to identify key intracellular mediators of syndecan-1 endocytosis.
METHODS/RESULTS Unclustered FcR-Synd1 and syndecan-1 each co-immunoprecipitated with tubulin, a binding partner for MKKK. About 15 min after ligand binding, FcR-Synd1 began to dissociate from tubulin. Tyrosine phosphorylation of FcR-Synd1 began ∼30min after clustering, at which time the chimera recruited cortactin, a molecule known to bridge to actin. Replacement of all four tyrosines in FcR-Synd1 with alanines still allowed raft localization and loss of tubulin upon clustering, but blocked phosphorylation, recruitment of cortactin, and efficient endocytosis. Knock-down of cortactin still allowed raft localization, tubulin loss, and tyrosine phosphorylation upon clustering of FcR-Synd1 or syndecan-1, but blocked their efficient endocytosis.
CONCLUSIONS These results establish a causal chain, in which clustering leads to raft localization, tubulin loss, and then MKKK-dependent tyrosine phosphorylation. Phosphorylation triggers the recruitment of cortactin, an essential mediator of efficient actin-dependent endocytosis. Our findings provide new insights to investigate the regulation of syndecan-mediated endocytosis in normal and diseased states and to assess its direct role in the catabolism of remnant lipoproteins and other ligands in vivo.
- © 2011 by American Heart Association, Inc.