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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

Hep endocytosis by cells expressing WT or HARE CD variants. Cells expressing WT (●), EV (○), or HARE CD mutants ((a) ΔM1 (Δ), ΔM2 (▼), ΔM3 (□), and ΔM4 (■) and (b) +M3 (Y2519A) (Δ), and WT (Y2519A) (▼)) were pretreated and incubated with 125I-SA•b-Hep complexes at 37°C as in Figure 2 for the indicated times to assess uptake rates as described in Methods section. Values are means ± SE (n = 9) and all linear regression lines had correlation coefficients ≥0.97.
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fig3: Hep endocytosis by cells expressing WT or HARE CD variants. Cells expressing WT (●), EV (○), or HARE CD mutants ((a) ΔM1 (Δ), ΔM2 (▼), ΔM3 (□), and ΔM4 (■) and (b) +M3 (Y2519A) (Δ), and WT (Y2519A) (▼)) were pretreated and incubated with 125I-SA•b-Hep complexes at 37°C as in Figure 2 for the indicated times to assess uptake rates as described in Methods section. Values are means ± SE (n = 9) and all linear regression lines had correlation coefficients ≥0.97.

Mentions: To understand further the importance of human HARE having the ability to internalize both HA and Hep, we wanted to determine which of the four CD endocytic motifs were functional for each ligand. We previously found that HARE expression levels, as well as HA binding to surface and intracellular HARE, were similar to WT in a panel of stable Flp-In 293 cell lines expressing various CD-mutants [26]. Here we used a set of variant cell lines, expressing HARE mutants that were either single-motif deletions or containing a single-motif (i.e., three motifs deleted). To determine whether the cellular HARE distribution of Hep binding was affected in any of the variants, we compared 125I-SA•b-Hep binding at 4°C to cell surface or total cellular HARE (cell surface and intracellular receptors) in the various HARE CD-mutant cells. Total and surface binding were monitored in the presence or absence of digitonin, respectively, under conditions that selectively permeabilize endocytic, but not nuclear, mitochondrial or lysosomal compartments [31, 39]. Since Hep nonspecifically binds to many cell surface and intracellular proteins, the binding of Hep by EV cells is higher relative to WT cells than the nonspecific binding of HA [13, 26]. Only small amounts of 125I-SA•biotin (e.g., <1% of 125I-SA•b-Hep values) bound to cells and this did not increase with time [13]. In contrast, 125I-SA•b-Hep uptake was time-dependent and linear over 4 h, as in Figure 3.


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 endocytosis by cells expressing WT or HARE CD variants. Cells expressing WT (●), EV (○), or HARE CD mutants ((a) ΔM1 (Δ), ΔM2 (▼), ΔM3 (□), and ΔM4 (■) and (b) +M3 (Y2519A) (Δ), and WT (Y2519A) (▼)) were pretreated and incubated with 125I-SA•b-Hep complexes at 37°C as in Figure 2 for the indicated times to assess uptake rates as described in Methods section. Values are means ± SE (n = 9) and all linear regression lines had correlation coefficients ≥0.97.
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Related In: Results  -  Collection

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fig3: Hep endocytosis by cells expressing WT or HARE CD variants. Cells expressing WT (●), EV (○), or HARE CD mutants ((a) ΔM1 (Δ), ΔM2 (▼), ΔM3 (□), and ΔM4 (■) and (b) +M3 (Y2519A) (Δ), and WT (Y2519A) (▼)) were pretreated and incubated with 125I-SA•b-Hep complexes at 37°C as in Figure 2 for the indicated times to assess uptake rates as described in Methods section. Values are means ± SE (n = 9) and all linear regression lines had correlation coefficients ≥0.97.
Mentions: To understand further the importance of human HARE having the ability to internalize both HA and Hep, we wanted to determine which of the four CD endocytic motifs were functional for each ligand. We previously found that HARE expression levels, as well as HA binding to surface and intracellular HARE, were similar to WT in a panel of stable Flp-In 293 cell lines expressing various CD-mutants [26]. Here we used a set of variant cell lines, expressing HARE mutants that were either single-motif deletions or containing a single-motif (i.e., three motifs deleted). To determine whether the cellular HARE distribution of Hep binding was affected in any of the variants, we compared 125I-SA•b-Hep binding at 4°C to cell surface or total cellular HARE (cell surface and intracellular receptors) in the various HARE CD-mutant cells. Total and surface binding were monitored in the presence or absence of digitonin, respectively, under conditions that selectively permeabilize endocytic, but not nuclear, mitochondrial or lysosomal compartments [31, 39]. Since Hep nonspecifically binds to many cell surface and intracellular proteins, the binding of Hep by EV cells is higher relative to WT cells than the nonspecific binding of HA [13, 26]. Only small amounts of 125I-SA•biotin (e.g., <1% of 125I-SA•b-Hep values) bound to cells and this did not increase with time [13]. In contrast, 125I-SA•b-Hep uptake was time-dependent and linear over 4 h, as in Figure 3.

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