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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 hyperosmolar conditions. Cells expressing EV, WT, or the indicated single-motif deletion HARE CD mutants were grown and pretreated as in Figure 2 and then preincubated at 37°C for 30 min with Endocytosis Medium with (white) or without (black) 0.45 M sucrose. The cells were then incubated with 125I-labelled HA (a) or Hep (b) at 37°C for 4 h and processed as described in Methods section. Values are means ± SE (n = 6) and significant differences (assessed by Student's t-test) between control and sucrose-treated samples are indicated: #P < 0.05; *P < 0.005; **P < 0.0005.
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fig5: Endocytosis of HA and Hep by HARE CD variants is blocked by hyperosmolar conditions. Cells expressing EV, WT, or the indicated single-motif deletion HARE CD mutants were grown and pretreated as in Figure 2 and then preincubated at 37°C for 30 min with Endocytosis Medium with (white) or without (black) 0.45 M sucrose. The cells were then incubated with 125I-labelled HA (a) or Hep (b) at 37°C for 4 h and processed as described in Methods section. Values are means ± SE (n = 6) and significant differences (assessed by Student's t-test) between control and sucrose-treated samples are indicated: #P < 0.05; *P < 0.005; **P < 0.0005.

Mentions: The unexpected behavior of HARE single-motif containing variants prompted us to verify that the various HARE CD variants mediate endocytosis using a clathrin coated pit pathway, as shown previously for native and recombinant WT HARE [28, 40, 45]. Hyperosmolar conditions inhibit clathrin assembly into coated pits and, thus, clathrin-dependent internalization of many plasma membrane receptors [46–48]. To verify further that ligand uptake by the various HARE CD-mutant cells is clathrin-dependent, we assessed the effects of hyperosmolarity on endocytosis using medium containing 0.45 M sucrose. Hyperosmolar sucrose treatment blocked internalization of HA by ~77% in WT cells compared to control (untreated) cells and by 40–70% in the single-motif deletion HARE CD mutants (Figure 5(a)). Similar results were obtained for the effects of hyperosmolarity on Hep uptake (Figure 5(b)). Overall, the results confirm that Hep and HA internalization by the various HARE CD mutants occurs via 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 hyperosmolar conditions. Cells expressing EV, WT, or the indicated single-motif deletion HARE CD mutants were grown and pretreated as in Figure 2 and then preincubated at 37°C for 30 min with Endocytosis Medium with (white) or without (black) 0.45 M sucrose. The cells were then incubated with 125I-labelled HA (a) or Hep (b) at 37°C for 4 h and processed as described in Methods section. Values are means ± SE (n = 6) and significant differences (assessed by Student's t-test) between control and sucrose-treated samples are indicated: #P < 0.05; *P < 0.005; **P < 0.0005.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig5: Endocytosis of HA and Hep by HARE CD variants is blocked by hyperosmolar conditions. Cells expressing EV, WT, or the indicated single-motif deletion HARE CD mutants were grown and pretreated as in Figure 2 and then preincubated at 37°C for 30 min with Endocytosis Medium with (white) or without (black) 0.45 M sucrose. The cells were then incubated with 125I-labelled HA (a) or Hep (b) at 37°C for 4 h and processed as described in Methods section. Values are means ± SE (n = 6) and significant differences (assessed by Student's t-test) between control and sucrose-treated samples are indicated: #P < 0.05; *P < 0.005; **P < 0.0005.
Mentions: The unexpected behavior of HARE single-motif containing variants prompted us to verify that the various HARE CD variants mediate endocytosis using a clathrin coated pit pathway, as shown previously for native and recombinant WT HARE [28, 40, 45]. Hyperosmolar conditions inhibit clathrin assembly into coated pits and, thus, clathrin-dependent internalization of many plasma membrane receptors [46–48]. To verify further that ligand uptake by the various HARE CD-mutant cells is clathrin-dependent, we assessed the effects of hyperosmolarity on endocytosis using medium containing 0.45 M sucrose. Hyperosmolar sucrose treatment blocked internalization of HA by ~77% in WT cells compared to control (untreated) cells and by 40–70% in the single-motif deletion HARE CD mutants (Figure 5(a)). Similar results were obtained for the effects of hyperosmolarity on Hep uptake (Figure 5(b)). Overall, the results confirm that Hep and HA internalization by the various HARE CD mutants occurs via 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