Abstract 3623: Molecular Determinants Of Endocytosis And Tyrosine Phosphorylation Within The Cytoplasmic Tail Of Human Syndecan-1, A Receptor For Remnant Lipoproteins
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-Synd, that consists of an IgG Fc receptor ectodomain linked to the transmembrane and cytoplasmic regions of syndecan-1, we found that efficient endocytosis is triggered by clustering of syndecan or the chimera. Syndecan clustering would be expected upon binding a multivalent ligand, such as a remnant lipoprotein. Clustering of the syndecan transmembrane and cytoplasmic domains causes their rapid movement into cholesterol-rich, detergent-insoluble, membrane rafts, and then the actual uptake into the cell requires recruitment of tyrosine kinases and the actin cytoskeleton (Biochem J 351:607, 2000). We now sought to identify the molecular determinants. We performed alanine scanning mutagenesis of 4 –5 consecutive residues at a time, to cover the entire syndecan cytoplasmic tail within the FcR-Synd chimera (excepting the first cytoplasmic residue, R, which was required for export to the cell surface). The parent and mutant constructs were each expressed in McArdle 7777 hepatoma cells. All alanine scanning mutants moved into rafts upon clustering. Replacement of a highly conserved, juxtamembrane motif, MKKK, with 4 alanines was the only mutation that blocked efficient endocytosis after clustering. Despite the presence of conserved lysyl residues, we did not find ubiquitinylated FcR-Synd without or with clustering. Clustering did, however, induce tyrosine phosphorylation of FcR-Synd, and this effect was absent from the mutant lacking the MKKK motif. Syndecan-mediated endocytosis was recently reported to activate extracellular signal-related kinases (ERK1/2), perhaps through the ectodomain (J Cell Biochem 99:936, 2006). Here, we found that engagement of the FcR-Synd chimera activated ERK, implicating the syndecan cytoplasmic tail. Furthermore, the ERK inhibitor U0126 blocked efficient endocytosis of FcR-Synd upon clustering. Overall, our results identify a novel motif, MKKK, in triggering tyrosine phosphorylation of the syndecan cytoplasmic tail and then endocytosis via rafts, in a process dependent on ERK signaling.