The Cytoplasmic Domain of the Hyaluronan Receptor for Endocytosis (HARE) Contains Multiple Endocytic Motifs Targeting Coated Pit-mediated Internalization

The hyaluronic acid (HA) receptor for endocytosis (HARE) is the primary scavenger receptor for HA and chondroitin sulfates in mammals. The two human isoforms of HARE (full-length 315-kDa and a 190-kDa proteolytic cleavage product), which are type I single-pass membrane proteins, are highly expressed in sinusoidal endothelial cells of lymph nodes, liver, and spleen. Their identical HARE cytoplasmic domains contain four candidate AP-2/clathrin-mediated endocytic signaling motifs as follows: YSYFRI2485, FQHF2495, NPLY2519, and DPF2534 (315-HARE numbering). Stably transfected cells expressing 190-HARE(ΔYSYFRI), 190-HARE(ΔFQHF), or 190-HARE(ΔNPLY) (lacking Motifs 1, 2, or 3) had decreased 125I-HA endocytosis rates of ~49, ~39, and ~56%, respectively (relative to wild type). In contrast, 190-HARE(ΔDPF) cells (lacking Motif 4) showed no change in HA endocytic rate. Deletions of motifs 1 and 2 or of 1, 2, and 4 decreased the rate of HA endocytosis by only ~41%. Endocytosis was ~95% decreased in mutants lacking all four motifs. Cells expressing a 190-HARE(Y2519A) mutant of the NPLY motif retained 85–90% of wild type endocytosis, whereas this mutation in the triple motif deletant decreased endocytosis to ~7% of wild type. Tyr in NPLY2519 is thus important for endocytosis. All HARE mutants showed similar HA binding and degradation of the internalized HA, indicating that altering endocytic motifs did not affect ectodomain binding of HA or targeting of internalized HA to lysosomes. We conclude that, although NPLY may be the most important motif, it functions together with two other endocytic motifs; thus three signal sequences (YSYFRI, FQHF, and NPLY) provide redundancy to mediate coated pit targeting and endocytosis of HARE