HARE-Mediated Endocytosis of Hyaluronan and Heparin Is Targeted by Different Subsets of Three Endocytic Motifs

Abstract EN

The hyaluronan (HA) receptor for endocytosis (HARE) is a multifunctional recycling clearancereceptor for 14 different ligands, including HA and heparin (Hep), which bind to discrete nonoverlappingsites.

Four different functional endocytic motifs (M) in the cytoplasmic domain(CD) target coated pit mediated uptake: (YSYFRI2485 (M1), FQHF2495 (M2), NPLY2519 (M3), andDPF2534 (M4)).

We previously found (Pandey et al.

J.

Biol.

Chem.

283, 21453, 2008) that M1,M2, and M3 mediate endocytosis of HA.

Here we assessed the ability of HARE variants with asingle-motif deletion or containing only a single motif to endocytose HA or Hep.

Single-motifdeletion variants lacking M1, M3, or M4 (a different subset than involved in HA uptake) showed decreased Hependocytosis, although M3 was the most active; the remaining redundant motifs did notcompensate for loss of other motifs.

Surprisingly, a HARE CD variant with only M3 internalizedboth HA and Hep, whereas variants with either M2 or M4 alone did not endocytose either ligand.

Internalization of HAand Hep by HARE CD mutants was dynamin-dependent and was inhibited byhyperosmolarity, confirming clathrin-mediated endocytosis.

The results indicate a complicatedrelationship among multiple CD motifs that target coated pit uptake and a more fundamental rolefor motif M3.