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

Pandey MS, Harris EN, Weigel PH - Int J Cell Biol (2015)

Bottom Line: We previously found (Pandey et al.Biol.Single-motif deletion variants lacking M1, M3, or M4 (a different subset than involved in HA uptake) showed decreased Hep endocytosis, although M3 was the most active; the remaining redundant motifs did not compensate for loss of other motifs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Biology, Penn State Hershey College of Medicine, Hershey, PA 17033, USA.

ABSTRACT
The hyaluronan (HA) receptor for endocytosis (HARE) is a multifunctional recycling clearance receptor for 14 different ligands, including HA and heparin (Hep), which bind to discrete nonoverlapping sites. Four different functional endocytic motifs (M) in the cytoplasmic domain (CD) target coated pit mediated uptake: (YSYFRI(2485) (M1), FQHF(2495) (M2), NPLY(2519) (M3), and DPF(2534) (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 a single-motif deletion or containing only a single motif to endocytose HA or Hep. Single-motif deletion variants lacking M1, M3, or M4 (a different subset than involved in HA uptake) showed decreased Hep endocytosis, although M3 was the most active; the remaining redundant motifs did not compensate for loss of other motifs. Surprisingly, a HARE CD variant with only M3 internalized both HA and Hep, whereas variants with either M2 or M4 alone did not endocytose either ligand. Internalization of HA and Hep by HARE CD mutants was dynamin-dependent and was inhibited by hyperosmolarity, confirming clathrin-mediated endocytosis. The results indicate a complicated relationship among multiple CD motifs that target coated pit uptake and a more fundamental role for motif M3.

No MeSH data available.


Different sets of HARE CD endocytic motifs are functional during HA versus Hep endocytosis. The four HARE endocytic motifs (boldface) examined are denoted by M1 (YSYFRI2485), M2 (FQHF2495), M3 (NPLY2519), and M4 (DPF2534). The two different subsets of three of these motifs active in the coated pit mediated endocytosis of HA (M1, M2, and M3) or Hep (M1, M3, and M4) are highlighted by brackets. M3 is highlighted in red to indicate its special significance as the only motif of the three we were able to test (M2, M3, and M4) that enabled HARE to target coated pit mediated endocytosis of both HA and Hep.
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Related In: Results  -  Collection


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fig7: Different sets of HARE CD endocytic motifs are functional during HA versus Hep endocytosis. The four HARE endocytic motifs (boldface) examined are denoted by M1 (YSYFRI2485), M2 (FQHF2495), M3 (NPLY2519), and M4 (DPF2534). The two different subsets of three of these motifs active in the coated pit mediated endocytosis of HA (M1, M2, and M3) or Hep (M1, M3, and M4) are highlighted by brackets. M3 is highlighted in red to indicate its special significance as the only motif of the three we were able to test (M2, M3, and M4) that enabled HARE to target coated pit mediated endocytosis of both HA and Hep.

Mentions: An unexpected finding in this study was that HARE utilizes a different subset of three motifs for the endocytosis of Hep compared to HA (Figure 7). Three of the four endocytic motifs in the HARE CD (M1 (YSYFRI2485), M3 (NPLY2519), and M4 (DPF2534)) are utilized for Hep internalization. In contrast, a different subset of three motifs (M1, M2 (FQHF2595), and M3) is utilized for HA endocytosis [26]. This result and the previous finding that Hep and HA bind to independent nonoverlapping sites in the HARE ectodomain [2] indicate that the binding of HA or Hep may create distinct conformational states within the intracellular CD that promoted differential recognition of endocytic motifs M2 and M4 by the relevant adaptor proteins. Different conformational or multimeric states of the intracellular CD could favor efficient binding of particular adaptor proteins to specific motifs. The CD conformation of HARE-HA complexes may allow M2 recognition by an appropriate adaptor protein, but not M4 recognition, whereas the CD conformation of HARE-Hep complexes may allow M4 recognition by an appropriate adaptor protein, but not M2 recognition. Consistent with the idea that binding in the ectodomain may influence intracellular signaling, Hep does not bind within the HA-binding HARE Link domain, whereas both HA and Hep bind to the Link domain of TSG6 [67].


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

Pandey MS, Harris EN, Weigel PH - Int J Cell Biol (2015)

Different sets of HARE CD endocytic motifs are functional during HA versus Hep endocytosis. The four HARE endocytic motifs (boldface) examined are denoted by M1 (YSYFRI2485), M2 (FQHF2495), M3 (NPLY2519), and M4 (DPF2534). The two different subsets of three of these motifs active in the coated pit mediated endocytosis of HA (M1, M2, and M3) or Hep (M1, M3, and M4) are highlighted by brackets. M3 is highlighted in red to indicate its special significance as the only motif of the three we were able to test (M2, M3, and M4) that enabled HARE to target coated pit mediated endocytosis of both HA and Hep.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4390207&req=5

fig7: Different sets of HARE CD endocytic motifs are functional during HA versus Hep endocytosis. The four HARE endocytic motifs (boldface) examined are denoted by M1 (YSYFRI2485), M2 (FQHF2495), M3 (NPLY2519), and M4 (DPF2534). The two different subsets of three of these motifs active in the coated pit mediated endocytosis of HA (M1, M2, and M3) or Hep (M1, M3, and M4) are highlighted by brackets. M3 is highlighted in red to indicate its special significance as the only motif of the three we were able to test (M2, M3, and M4) that enabled HARE to target coated pit mediated endocytosis of both HA and Hep.
Mentions: An unexpected finding in this study was that HARE utilizes a different subset of three motifs for the endocytosis of Hep compared to HA (Figure 7). Three of the four endocytic motifs in the HARE CD (M1 (YSYFRI2485), M3 (NPLY2519), and M4 (DPF2534)) are utilized for Hep internalization. In contrast, a different subset of three motifs (M1, M2 (FQHF2595), and M3) is utilized for HA endocytosis [26]. This result and the previous finding that Hep and HA bind to independent nonoverlapping sites in the HARE ectodomain [2] indicate that the binding of HA or Hep may create distinct conformational states within the intracellular CD that promoted differential recognition of endocytic motifs M2 and M4 by the relevant adaptor proteins. Different conformational or multimeric states of the intracellular CD could favor efficient binding of particular adaptor proteins to specific motifs. The CD conformation of HARE-HA complexes may allow M2 recognition by an appropriate adaptor protein, but not M4 recognition, whereas the CD conformation of HARE-Hep complexes may allow M4 recognition by an appropriate adaptor protein, but not M2 recognition. Consistent with the idea that binding in the ectodomain may influence intracellular signaling, Hep does not bind within the HA-binding HARE Link domain, whereas both HA and Hep bind to the Link domain of TSG6 [67].

Bottom Line: We previously found (Pandey et al.Biol.Single-motif deletion variants lacking M1, M3, or M4 (a different subset than involved in HA uptake) showed decreased Hep endocytosis, although M3 was the most active; the remaining redundant motifs did not compensate for loss of other motifs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Biology, Penn State Hershey College of Medicine, Hershey, PA 17033, USA.

ABSTRACT
The hyaluronan (HA) receptor for endocytosis (HARE) is a multifunctional recycling clearance receptor for 14 different ligands, including HA and heparin (Hep), which bind to discrete nonoverlapping sites. Four different functional endocytic motifs (M) in the cytoplasmic domain (CD) target coated pit mediated uptake: (YSYFRI(2485) (M1), FQHF(2495) (M2), NPLY(2519) (M3), and DPF(2534) (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 a single-motif deletion or containing only a single motif to endocytose HA or Hep. Single-motif deletion variants lacking M1, M3, or M4 (a different subset than involved in HA uptake) showed decreased Hep endocytosis, although M3 was the most active; the remaining redundant motifs did not compensate for loss of other motifs. Surprisingly, a HARE CD variant with only M3 internalized both HA and Hep, whereas variants with either M2 or M4 alone did not endocytose either ligand. Internalization of HA and Hep by HARE CD mutants was dynamin-dependent and was inhibited by hyperosmolarity, confirming clathrin-mediated endocytosis. The results indicate a complicated relationship among multiple CD motifs that target coated pit uptake and a more fundamental role for motif M3.

No MeSH data available.