Tyrosine sulfation of chemokine receptor CCR2 enhances interactions with both monomeric and dimeric forms of the chemokine monocyte chemoattractant protein-1 (MCP-1)

Joshua Tan Hoong Yu, Justin Peter Ludeman, Jamie Wedderburn, Meritxell Canals, Pamela Heidi Hall, Stephen J Butler, Deni Taleski, Arthur Christopoulos, Michael John Hickey, Richard James Payne, Martin J Stone

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Chemokine receptors are commonly post-translationally sulfated on tyrosine residues in their N-terminal regions, the initial site of binding to chemokine ligands. We have investigated the effect of tyrosine sulfation of the chemokine receptor CCR2 on its interactions with the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Inhibition of CCR2 sulfation, by growth of expressing cells in the presence of sodium chlorate, significantly reduced the potency for MCP-1 activation of CCR2. MCP-1 exists in equilibrium between monomeric and dimeric forms. The obligate monomeric mutant MCP-1(P8A) was similar to wild type MCP-1 in its ability to induce leukocyte recruitment in vivo, whereas the obligate dimeric mutant MCP-1 (T10C) was less effective at inducing leukocyte recruitment in vivo. In 2D NMR experiments, sulfated peptides derived from the N-terminal region of CCR2 bound to both the monomeric and dimeric forms of wild type MCP-1 and shifted the equilibrium to favour the monomeric form. Similarly, MCP-1(P8A) bound more tightly than MCP-1(T10C) to the CCR2-derived sulfopeptides. NMR chemical shift mapping using the MCP-1 mutants showed that the sulfated N-terminal region of CCR2 binds to the same region (N-loop and beta3-strand) of both monomeric and dimeric MCP-1 but that binding to the dimeric form also influences the environment of chemokine N-terminal residues, which are involved in dimer formation. We conclude that interaction with the sulfated N-terminus of CCR2 destabilises the dimerization interface of inactive dimeric MCP-1, thus inducing dissociation to the active monomeric state.
Original languageEnglish
Pages (from-to)10024 - 10034
Number of pages11
JournalJournal of Biological Chemistry
Volume288
Issue number14
DOIs
Publication statusPublished - 2013

Cite this

Tan Hoong Yu, Joshua ; Ludeman, Justin Peter ; Wedderburn, Jamie ; Canals, Meritxell ; Hall, Pamela Heidi ; Butler, Stephen J ; Taleski, Deni ; Christopoulos, Arthur ; Hickey, Michael John ; Payne, Richard James ; Stone, Martin J. / Tyrosine sulfation of chemokine receptor CCR2 enhances interactions with both monomeric and dimeric forms of the chemokine monocyte chemoattractant protein-1 (MCP-1). In: Journal of Biological Chemistry. 2013 ; Vol. 288, No. 14. pp. 10024 - 10034.
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abstract = "Chemokine receptors are commonly post-translationally sulfated on tyrosine residues in their N-terminal regions, the initial site of binding to chemokine ligands. We have investigated the effect of tyrosine sulfation of the chemokine receptor CCR2 on its interactions with the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Inhibition of CCR2 sulfation, by growth of expressing cells in the presence of sodium chlorate, significantly reduced the potency for MCP-1 activation of CCR2. MCP-1 exists in equilibrium between monomeric and dimeric forms. The obligate monomeric mutant MCP-1(P8A) was similar to wild type MCP-1 in its ability to induce leukocyte recruitment in vivo, whereas the obligate dimeric mutant MCP-1 (T10C) was less effective at inducing leukocyte recruitment in vivo. In 2D NMR experiments, sulfated peptides derived from the N-terminal region of CCR2 bound to both the monomeric and dimeric forms of wild type MCP-1 and shifted the equilibrium to favour the monomeric form. Similarly, MCP-1(P8A) bound more tightly than MCP-1(T10C) to the CCR2-derived sulfopeptides. NMR chemical shift mapping using the MCP-1 mutants showed that the sulfated N-terminal region of CCR2 binds to the same region (N-loop and beta3-strand) of both monomeric and dimeric MCP-1 but that binding to the dimeric form also influences the environment of chemokine N-terminal residues, which are involved in dimer formation. We conclude that interaction with the sulfated N-terminus of CCR2 destabilises the dimerization interface of inactive dimeric MCP-1, thus inducing dissociation to the active monomeric state.",
author = "{Tan Hoong Yu}, Joshua and Ludeman, {Justin Peter} and Jamie Wedderburn and Meritxell Canals and Hall, {Pamela Heidi} and Butler, {Stephen J} and Deni Taleski and Arthur Christopoulos and Hickey, {Michael John} and Payne, {Richard James} and Stone, {Martin J}",
year = "2013",
doi = "10.1074/jbc.M112.447359",
language = "English",
volume = "288",
pages = "10024 -- 10034",
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publisher = "American Society for Biochemistry and Molecular Biology",
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Tyrosine sulfation of chemokine receptor CCR2 enhances interactions with both monomeric and dimeric forms of the chemokine monocyte chemoattractant protein-1 (MCP-1). / Tan Hoong Yu, Joshua; Ludeman, Justin Peter; Wedderburn, Jamie; Canals, Meritxell; Hall, Pamela Heidi; Butler, Stephen J; Taleski, Deni; Christopoulos, Arthur; Hickey, Michael John; Payne, Richard James; Stone, Martin J.

In: Journal of Biological Chemistry, Vol. 288, No. 14, 2013, p. 10024 - 10034.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Tyrosine sulfation of chemokine receptor CCR2 enhances interactions with both monomeric and dimeric forms of the chemokine monocyte chemoattractant protein-1 (MCP-1)

AU - Tan Hoong Yu, Joshua

AU - Ludeman, Justin Peter

AU - Wedderburn, Jamie

AU - Canals, Meritxell

AU - Hall, Pamela Heidi

AU - Butler, Stephen J

AU - Taleski, Deni

AU - Christopoulos, Arthur

AU - Hickey, Michael John

AU - Payne, Richard James

AU - Stone, Martin J

PY - 2013

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N2 - Chemokine receptors are commonly post-translationally sulfated on tyrosine residues in their N-terminal regions, the initial site of binding to chemokine ligands. We have investigated the effect of tyrosine sulfation of the chemokine receptor CCR2 on its interactions with the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Inhibition of CCR2 sulfation, by growth of expressing cells in the presence of sodium chlorate, significantly reduced the potency for MCP-1 activation of CCR2. MCP-1 exists in equilibrium between monomeric and dimeric forms. The obligate monomeric mutant MCP-1(P8A) was similar to wild type MCP-1 in its ability to induce leukocyte recruitment in vivo, whereas the obligate dimeric mutant MCP-1 (T10C) was less effective at inducing leukocyte recruitment in vivo. In 2D NMR experiments, sulfated peptides derived from the N-terminal region of CCR2 bound to both the monomeric and dimeric forms of wild type MCP-1 and shifted the equilibrium to favour the monomeric form. Similarly, MCP-1(P8A) bound more tightly than MCP-1(T10C) to the CCR2-derived sulfopeptides. NMR chemical shift mapping using the MCP-1 mutants showed that the sulfated N-terminal region of CCR2 binds to the same region (N-loop and beta3-strand) of both monomeric and dimeric MCP-1 but that binding to the dimeric form also influences the environment of chemokine N-terminal residues, which are involved in dimer formation. We conclude that interaction with the sulfated N-terminus of CCR2 destabilises the dimerization interface of inactive dimeric MCP-1, thus inducing dissociation to the active monomeric state.

AB - Chemokine receptors are commonly post-translationally sulfated on tyrosine residues in their N-terminal regions, the initial site of binding to chemokine ligands. We have investigated the effect of tyrosine sulfation of the chemokine receptor CCR2 on its interactions with the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Inhibition of CCR2 sulfation, by growth of expressing cells in the presence of sodium chlorate, significantly reduced the potency for MCP-1 activation of CCR2. MCP-1 exists in equilibrium between monomeric and dimeric forms. The obligate monomeric mutant MCP-1(P8A) was similar to wild type MCP-1 in its ability to induce leukocyte recruitment in vivo, whereas the obligate dimeric mutant MCP-1 (T10C) was less effective at inducing leukocyte recruitment in vivo. In 2D NMR experiments, sulfated peptides derived from the N-terminal region of CCR2 bound to both the monomeric and dimeric forms of wild type MCP-1 and shifted the equilibrium to favour the monomeric form. Similarly, MCP-1(P8A) bound more tightly than MCP-1(T10C) to the CCR2-derived sulfopeptides. NMR chemical shift mapping using the MCP-1 mutants showed that the sulfated N-terminal region of CCR2 binds to the same region (N-loop and beta3-strand) of both monomeric and dimeric MCP-1 but that binding to the dimeric form also influences the environment of chemokine N-terminal residues, which are involved in dimer formation. We conclude that interaction with the sulfated N-terminus of CCR2 destabilises the dimerization interface of inactive dimeric MCP-1, thus inducing dissociation to the active monomeric state.

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