Transmembrane and cytoplasmic domains of syndecan mediate a multi-step endocytic pathway involving detergent-insoluble membrane rafts

Syndecan heparan sulphate proteoglycans directly mediate a novel endocytic pathway. Using Chinese hamster ovary cells expressing the human syndecan 1 core protein or a chimaeric receptor, FcR-Synd, consisting of the ectodomai n of the IgG Fc receptor Ia linked to the transmembrane and cytoplasmic dom ains of syndecan 1, we previously reported that efficient internalization i s triggered by ligand clustering, requires intact actin microfilaments and tyrosine kinases, proceeds with a t(1/2) of approx. 1 h and is distinct fro m coated-pit pathways. We have now examined the involvement of cholesterol- rich, detergent-insoluble membrane rafts. On clustering, I-125-labelled IgG bound to FcR-Synd rapidly became insoluble in cold Triton X-100, well befo re endocytosis, Insolubility of clustered FcR-Synd ligand did not require t he syndecan ectodomain, linkage of the cytoplasmic tail to the cytoskeleton , or energy-dependent cellular metabolism. Pretreatment of cells with cyclo dextrin to deplete cholesterol from rafts abolished insolubility of the clu stered ligand and inhibited endocytosis in a dose-responsive fashion. Simil ar results were obtained with I-125-labelled lipoprotein lipase bound to au thentic cell-surface syndecan. In contrast, the 39 kDa receptor-associated protein (RAP), a coated-pit ligand, was more than 80% soluble in cold Trito n even after internalization; cellular cholesterol depletion failed to subs tantially affect the internalization of I-125-RAP. Overall, our results ind icate a multi-step endocytic process consisting of ligand binding, clusteri ng, energy-independent lateral movement into detergent-insoluble membrane r afts and finally recruitment of actin and tyrosine kinases to bring the lig ands into the cell.