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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.


HARE CD mutants with different combinations of the four endocytic motifs. The diagram illustrates the various combinations of HARE CD motifs (M1, M2, M3, and M4) present (dark gray boxes) or deleted (light gray boxes with X) in the panel of stable HARE-CD variant cell lines used here. The single transmembrane domain (TMD, black box), C-terminal region (CT), and presence of the site-specific Y2519A mutation in M3 are indicated.
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fig1: HARE CD mutants with different combinations of the four endocytic motifs. The diagram illustrates the various combinations of HARE CD motifs (M1, M2, M3, and M4) present (dark gray boxes) or deleted (light gray boxes with X) in the panel of stable HARE-CD variant cell lines used here. The single transmembrane domain (TMD, black box), C-terminal region (CT), and presence of the site-specific Y2519A mutation in M3 are indicated.

Mentions: HARE and Stab2 are scavenger receptors that bind and clear 14 different ligands, including seven glycosaminoglycans, from lymph and blood. We designate the full-length 315 kDa protein as Stab2 and HARE as the 190 kDa isoform generated by an unknown proteolytic mechanism [33]. Both HARE/Stab2 are the main systemic clearance receptors for HA and presumably Hep, in all mammals studied [34–37]. HARE is the predominant Stab2-related protein expressed in sinusoidal endothelial cells of lymph node and liver, the main systemic clearance tissues [10, 38]. Although both HA and Hep are anionic glycosaminoglycans, they bind to discrete and nonoverlapping sites in the HARE ectodomain [2]. HA binding requires the Link domain, which it likely binds to directly, whereas Hep binds to an uncharacterized site and binding is unaffected by deletion of the Link domain [2]. Since, HA and Hep bind to different sites, we wanted to determine if HARE utilizes the same subset of three redundantly functional endocytic motifs for Hep endocytosis as found previously for HA endocytosis [26]. Most of the CD mutants used here had been characterized previously for their HARE-mediated HA binding and uptake ability. Two additional single-motif containing CD mutants were created for the present study (+M2 and +M4) to obtain a set of HARE CD variants expressing only one of the four motifs (e.g., ΔM1M2M4 = +M3); the panel of CD mutants used is shown schematically in Figure 1. We were not successful in creating cell lines expressing only motif M1.


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)

HARE CD mutants with different combinations of the four endocytic motifs. The diagram illustrates the various combinations of HARE CD motifs (M1, M2, M3, and M4) present (dark gray boxes) or deleted (light gray boxes with X) in the panel of stable HARE-CD variant cell lines used here. The single transmembrane domain (TMD, black box), C-terminal region (CT), and presence of the site-specific Y2519A mutation in M3 are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4390207&req=5

fig1: HARE CD mutants with different combinations of the four endocytic motifs. The diagram illustrates the various combinations of HARE CD motifs (M1, M2, M3, and M4) present (dark gray boxes) or deleted (light gray boxes with X) in the panel of stable HARE-CD variant cell lines used here. The single transmembrane domain (TMD, black box), C-terminal region (CT), and presence of the site-specific Y2519A mutation in M3 are indicated.
Mentions: HARE and Stab2 are scavenger receptors that bind and clear 14 different ligands, including seven glycosaminoglycans, from lymph and blood. We designate the full-length 315 kDa protein as Stab2 and HARE as the 190 kDa isoform generated by an unknown proteolytic mechanism [33]. Both HARE/Stab2 are the main systemic clearance receptors for HA and presumably Hep, in all mammals studied [34–37]. HARE is the predominant Stab2-related protein expressed in sinusoidal endothelial cells of lymph node and liver, the main systemic clearance tissues [10, 38]. Although both HA and Hep are anionic glycosaminoglycans, they bind to discrete and nonoverlapping sites in the HARE ectodomain [2]. HA binding requires the Link domain, which it likely binds to directly, whereas Hep binds to an uncharacterized site and binding is unaffected by deletion of the Link domain [2]. Since, HA and Hep bind to different sites, we wanted to determine if HARE utilizes the same subset of three redundantly functional endocytic motifs for Hep endocytosis as found previously for HA endocytosis [26]. Most of the CD mutants used here had been characterized previously for their HARE-mediated HA binding and uptake ability. Two additional single-motif containing CD mutants were created for the present study (+M2 and +M4) to obtain a set of HARE CD variants expressing only one of the four motifs (e.g., ΔM1M2M4 = +M3); the panel of CD mutants used is shown schematically in Figure 1. We were not successful in creating cell lines expressing only motif M1.

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.