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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.


Hep binding to cell surface and total HARE in CD variants. Cells expressing human HARE (WT), the indicated HARE CD mutants, or EV were grown, washed, and preincubated in Endocytosis Medium at 37°C for 1 h to allow clearance of serum-derived glycosaminoglycans bound to HARE. Cells were chilled to 4°C, washed, and incubated with 125I-SA•b-Hep complexes at 4°C and processed as described in Methods section to determine cell surface (a, c, e) or total cellular (b, d, f) specific 125I-SA•b-Hep binding. Values are means ± SE (n = 6–9) and significant differences (assessed by Student's t-test) between WT and a HARE CD variant are indicated: #P < 0.05.
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fig2: Hep binding to cell surface and total HARE in CD variants. Cells expressing human HARE (WT), the indicated HARE CD mutants, or EV were grown, washed, and preincubated in Endocytosis Medium at 37°C for 1 h to allow clearance of serum-derived glycosaminoglycans bound to HARE. Cells were chilled to 4°C, washed, and incubated with 125I-SA•b-Hep complexes at 4°C and processed as described in Methods section to determine cell surface (a, c, e) or total cellular (b, d, f) specific 125I-SA•b-Hep binding. Values are means ± SE (n = 6–9) and significant differences (assessed by Student's t-test) between WT and a HARE CD variant are indicated: #P < 0.05.

Mentions: Cell surface (Figures 2(a), 2(c), and 2(e)) and total (Figures 2(b), 2(d), and 2(f)) 125I-SA•b-Hep binding to WT or HARE CD-mutant cells were 2-3 times greater than to EV cells.As expected, the distribution of 125I-SA•b-Hep binding sites between surface and internal was similar to that for HA binding in WT and the HARE CD mutants [26]. HARE is a constitutively active receptor involved in continuous and repeated cycles of ligand internalization and the HARE recycling time of 7–9 min [28, 40] is similar to that of other constitutively active recycling receptors [13, 41, 42]. The majority of recycling receptors, including HARE [28, 33], are localized in intracellular endocytic and recycling compartments. Thus, Hep total binding (surface and internal) by WT or CD-mutant cells was much greater than surface binding, as expected. Among the group of nine CD-mutants examined, there were no significant differences in Hep surface binding (Figure 2 top panels), confirming that deletion of one or more endocytic motifs did not alter the dynamic ongoing movement of HARE to and from the cell surface; the steady-state surface receptor pool was similar among a set of HARE variants. Total Hep binding was identical to WT among the set of nine HARE mutants except for ΔM1 and ΔM3 (Figure 2(b)), which were significantly higher (P < 0.05).


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)

Hep binding to cell surface and total HARE in CD variants. Cells expressing human HARE (WT), the indicated HARE CD mutants, or EV were grown, washed, and preincubated in Endocytosis Medium at 37°C for 1 h to allow clearance of serum-derived glycosaminoglycans bound to HARE. Cells were chilled to 4°C, washed, and incubated with 125I-SA•b-Hep complexes at 4°C and processed as described in Methods section to determine cell surface (a, c, e) or total cellular (b, d, f) specific 125I-SA•b-Hep binding. Values are means ± SE (n = 6–9) and significant differences (assessed by Student's t-test) between WT and a HARE CD variant are indicated: #P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2: Hep binding to cell surface and total HARE in CD variants. Cells expressing human HARE (WT), the indicated HARE CD mutants, or EV were grown, washed, and preincubated in Endocytosis Medium at 37°C for 1 h to allow clearance of serum-derived glycosaminoglycans bound to HARE. Cells were chilled to 4°C, washed, and incubated with 125I-SA•b-Hep complexes at 4°C and processed as described in Methods section to determine cell surface (a, c, e) or total cellular (b, d, f) specific 125I-SA•b-Hep binding. Values are means ± SE (n = 6–9) and significant differences (assessed by Student's t-test) between WT and a HARE CD variant are indicated: #P < 0.05.
Mentions: Cell surface (Figures 2(a), 2(c), and 2(e)) and total (Figures 2(b), 2(d), and 2(f)) 125I-SA•b-Hep binding to WT or HARE CD-mutant cells were 2-3 times greater than to EV cells.As expected, the distribution of 125I-SA•b-Hep binding sites between surface and internal was similar to that for HA binding in WT and the HARE CD mutants [26]. HARE is a constitutively active receptor involved in continuous and repeated cycles of ligand internalization and the HARE recycling time of 7–9 min [28, 40] is similar to that of other constitutively active recycling receptors [13, 41, 42]. The majority of recycling receptors, including HARE [28, 33], are localized in intracellular endocytic and recycling compartments. Thus, Hep total binding (surface and internal) by WT or CD-mutant cells was much greater than surface binding, as expected. Among the group of nine CD-mutants examined, there were no significant differences in Hep surface binding (Figure 2 top panels), confirming that deletion of one or more endocytic motifs did not alter the dynamic ongoing movement of HARE to and from the cell surface; the steady-state surface receptor pool was similar among a set of HARE variants. Total Hep binding was identical to WT among the set of nine HARE mutants except for ΔM1 and ΔM3 (Figure 2(b)), which were significantly higher (P < 0.05).

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.