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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.


Related in: MedlinePlus

Endocytosis of HA and Hep by HARE CD variants is blocked by a dynamin inhibitor. WT cells were washed and preincubated in Endocytosis Medium as in Figure 2 and pretreated in medium with DMSO alone (black) or with 300 μM dynasore (white) at 37°C for 30 min. The medium was then replaced with fresh media containing DMSO alone or dynasore and 125I-labelled HA (a) or Hep (b). The cells were incubated at 37°C for 4 h and specific cell-associated ligand was determined as noted in Methods section. Values are the means ± SE (n = 3) and significant differences (Student's t-test) between treated and control samples are indicated: *P < 0.005; **P < 0.0005.
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fig6: Endocytosis of HA and Hep by HARE CD variants is blocked by a dynamin inhibitor. WT cells were washed and preincubated in Endocytosis Medium as in Figure 2 and pretreated in medium with DMSO alone (black) or with 300 μM dynasore (white) at 37°C for 30 min. The medium was then replaced with fresh media containing DMSO alone or dynasore and 125I-labelled HA (a) or Hep (b). The cells were incubated at 37°C for 4 h and specific cell-associated ligand was determined as noted in Methods section. Values are the means ± SE (n = 3) and significant differences (Student's t-test) between treated and control samples are indicated: *P < 0.005; **P < 0.0005.

Mentions: Many endocytic pathways in mammalian cells, including those involving clathrin-coated pits, phagocytosis, and caveolae, require the molecular motor protein dynamin for vesicle formation [49]. Dynasore is a small cell-permeable chemical that specifically inhibits the GTPase activity of dynamin and interferes with dynamin-dependent endocytic pathways [50]. Although not absolutely specific for clathrin-mediated uptake, dynasore should inhibit HARE-mediated uptake that occurs via coated pits. Dynasore treatment significantly inhibited HA uptake by HARE CD variants with alterations in the motif subset involved in HA uptake, compared to DMSO-alone controls (Figure 6(a)). Similarly, dynasore inhibited Hep endocytosis by ~70–85% in several HARE CD mutants of the motif subset involved in Hep uptake (Figure 6(b)). As expected, the above dynasore and hyperosmolar sensitivity results indicate that the various HARE CD mutants mediate HARE-Hep and HARE-HA endocytosis by dynamin-dependent clathrin-coated pit pathways.


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)

Endocytosis of HA and Hep by HARE CD variants is blocked by a dynamin inhibitor. WT cells were washed and preincubated in Endocytosis Medium as in Figure 2 and pretreated in medium with DMSO alone (black) or with 300 μM dynasore (white) at 37°C for 30 min. The medium was then replaced with fresh media containing DMSO alone or dynasore and 125I-labelled HA (a) or Hep (b). The cells were incubated at 37°C for 4 h and specific cell-associated ligand was determined as noted in Methods section. Values are the means ± SE (n = 3) and significant differences (Student's t-test) between treated and control samples are indicated: *P < 0.005; **P < 0.0005.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig6: Endocytosis of HA and Hep by HARE CD variants is blocked by a dynamin inhibitor. WT cells were washed and preincubated in Endocytosis Medium as in Figure 2 and pretreated in medium with DMSO alone (black) or with 300 μM dynasore (white) at 37°C for 30 min. The medium was then replaced with fresh media containing DMSO alone or dynasore and 125I-labelled HA (a) or Hep (b). The cells were incubated at 37°C for 4 h and specific cell-associated ligand was determined as noted in Methods section. Values are the means ± SE (n = 3) and significant differences (Student's t-test) between treated and control samples are indicated: *P < 0.005; **P < 0.0005.
Mentions: Many endocytic pathways in mammalian cells, including those involving clathrin-coated pits, phagocytosis, and caveolae, require the molecular motor protein dynamin for vesicle formation [49]. Dynasore is a small cell-permeable chemical that specifically inhibits the GTPase activity of dynamin and interferes with dynamin-dependent endocytic pathways [50]. Although not absolutely specific for clathrin-mediated uptake, dynasore should inhibit HARE-mediated uptake that occurs via coated pits. Dynasore treatment significantly inhibited HA uptake by HARE CD variants with alterations in the motif subset involved in HA uptake, compared to DMSO-alone controls (Figure 6(a)). Similarly, dynasore inhibited Hep endocytosis by ~70–85% in several HARE CD mutants of the motif subset involved in Hep uptake (Figure 6(b)). As expected, the above dynasore and hyperosmolar sensitivity results indicate that the various HARE CD mutants mediate HARE-Hep and HARE-HA endocytosis by dynamin-dependent clathrin-coated pit pathways.

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.


Related in: MedlinePlus