Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding

Line Barington; Pia C Rummel; Michael Lückmann; Heidi Pihl; Olav Larsen; Viktorija Daugvilaite; Anders H. Johnsen; Thomas M Frimurer; Stefanie Karlshøj; Mette M Rosenkilde

doi:10.1074/jbc.m115.706747

Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding

Line Barington, Pia C Rummel, Michael Lückmann, Heidi Pihl, Olav Larsen, Viktorija Daugvilaite, Anders H. Johnsen, Thomas M Frimurer, Stefanie Karlshøj, Mette M Rosenkilde

Department of Neuroscience

8 Citations (Scopus)

Abstract

Chemokine receptors play important roles in the immune system and are linked to several human diseases. The initial contact of chemokines with their receptors depends on highly specified extracellular receptor features. Here we investigate the importance of conserved extracellular disulfide bridges and aromatic residues in extracellular loop 2 (ECL-2) for ligand binding and activation in the chemokine receptor CCR8. We used inositol 1,4,5-trisphosphate accumulation and radioligand binding experiments to determine the impact of receptor mutagenesis on both chemokine and small molecule agonist and antagonist binding and action in CCR8. We find that the seven-transmembrane (TM) receptor conserved disulfide bridge (7TM bridge) linking transmembrane helix III (TMIII) and ECL-2 is crucial for chemokine and small molecule action, whereas the chemokine receptor conserved disulfide bridge between the N terminus and TMVII is needed only for chemokines. Furthermore, we find that two distinct aromatic residues in ECL-2, Tyr¹⁸⁴ (Cys + 1) and Tyr¹⁸⁷ (Cys + 4), are crucial for binding of the CC chemokines CCL1 (agonist) and MC148 (antagonist), respectively, but not for small molecule binding. Finally, using in silico modeling, we predict an aromatic cluster of interaction partners for Tyr¹⁸⁷ in TMIV (Phe¹⁷¹) and TMV (Trp¹⁹⁴). We show in vitro that these residues are crucial for the binding and action of MC148, thus supporting their participation in an aromatic cluster with Tyr¹⁸⁷. This aromatic cluster appears to be present in a large number of CC chemokine receptors and thereby could play a more general role to be exploited in future drug development targeting these receptors.

Original language	English
Journal	The Journal of Biological Chemistry
Volume	291
Issue number	31
Pages (from-to)	16208-16220
ISSN	0021-9258
DOIs	https://doi.org/10.1074/jbc.m115.706747
Publication status	Published - 29 Jul 2016

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Barington, L., Rummel, P. C., Lückmann, M., Pihl, H., Larsen, O., Daugvilaite, V., Johnsen, A. H., Frimurer, T. M., Karlshøj, S., & Rosenkilde, M. M. (2016). Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding. The Journal of Biological Chemistry, 291(31), 16208-16220. https://doi.org/10.1074/jbc.m115.706747

Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding. / Barington, Line; Rummel, Pia C; Lückmann, Michael et al.

In: The Journal of Biological Chemistry, Vol. 291, No. 31, 29.07.2016, p. 16208-16220.

Research output: Contribution to journal › Journal article › Research › peer-review

Barington, L, Rummel, PC, Lückmann, M, Pihl, H, Larsen, O , Daugvilaite, V, Johnsen, AH, Frimurer, TM, Karlshøj, S & Rosenkilde, MM 2016, 'Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding', The Journal of Biological Chemistry, vol. 291, no. 31, pp. 16208-16220. https://doi.org/10.1074/jbc.m115.706747

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title = "Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding",

abstract = "Chemokine receptors play important roles in the immune system and are linked to several human diseases. The initial contact of chemokines with their receptors depends on highly specified extracellular receptor features. Here we investigate the importance of conserved extracellular disulfide bridges and aromatic residues in extracellular loop 2 (ECL-2) for ligand binding and activation in the chemokine receptor CCR8. We used inositol 1,4,5-trisphosphate accumulation and radioligand binding experiments to determine the impact of receptor mutagenesis on both chemokine and small molecule agonist and antagonist binding and action in CCR8. We find that the seven-transmembrane (TM) receptor conserved disulfide bridge (7TM bridge) linking transmembrane helix III (TMIII) and ECL-2 is crucial for chemokine and small molecule action, whereas the chemokine receptor conserved disulfide bridge between the N terminus and TMVII is needed only for chemokines. Furthermore, we find that two distinct aromatic residues in ECL-2, Tyr184 (Cys + 1) and Tyr187 (Cys + 4), are crucial for binding of the CC chemokines CCL1 (agonist) and MC148 (antagonist), respectively, but not for small molecule binding. Finally, using in silico modeling, we predict an aromatic cluster of interaction partners for Tyr187 in TMIV (Phe171) and TMV (Trp194). We show in vitro that these residues are crucial for the binding and action of MC148, thus supporting their participation in an aromatic cluster with Tyr187. This aromatic cluster appears to be present in a large number of CC chemokine receptors and thereby could play a more general role to be exploited in future drug development targeting these receptors.",

author = "Line Barington and Rummel, {Pia C} and Michael L{\"u}ckmann and Heidi Pihl and Olav Larsen and Viktorija Daugvilaite and Johnsen, {Anders H.} and Frimurer, {Thomas M} and Stefanie Karlsh{\o}j and Rosenkilde, {Mette M}",

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T1 - Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding

AU - Barington, Line

AU - Rummel, Pia C

AU - Lückmann, Michael

AU - Pihl, Heidi

AU - Larsen, Olav

AU - Daugvilaite, Viktorija

AU - Johnsen, Anders H.

AU - Frimurer, Thomas M

AU - Karlshøj, Stefanie

AU - Rosenkilde, Mette M

PY - 2016/7/29

Y1 - 2016/7/29

N2 - Chemokine receptors play important roles in the immune system and are linked to several human diseases. The initial contact of chemokines with their receptors depends on highly specified extracellular receptor features. Here we investigate the importance of conserved extracellular disulfide bridges and aromatic residues in extracellular loop 2 (ECL-2) for ligand binding and activation in the chemokine receptor CCR8. We used inositol 1,4,5-trisphosphate accumulation and radioligand binding experiments to determine the impact of receptor mutagenesis on both chemokine and small molecule agonist and antagonist binding and action in CCR8. We find that the seven-transmembrane (TM) receptor conserved disulfide bridge (7TM bridge) linking transmembrane helix III (TMIII) and ECL-2 is crucial for chemokine and small molecule action, whereas the chemokine receptor conserved disulfide bridge between the N terminus and TMVII is needed only for chemokines. Furthermore, we find that two distinct aromatic residues in ECL-2, Tyr184 (Cys + 1) and Tyr187 (Cys + 4), are crucial for binding of the CC chemokines CCL1 (agonist) and MC148 (antagonist), respectively, but not for small molecule binding. Finally, using in silico modeling, we predict an aromatic cluster of interaction partners for Tyr187 in TMIV (Phe171) and TMV (Trp194). We show in vitro that these residues are crucial for the binding and action of MC148, thus supporting their participation in an aromatic cluster with Tyr187. This aromatic cluster appears to be present in a large number of CC chemokine receptors and thereby could play a more general role to be exploited in future drug development targeting these receptors.

AB - Chemokine receptors play important roles in the immune system and are linked to several human diseases. The initial contact of chemokines with their receptors depends on highly specified extracellular receptor features. Here we investigate the importance of conserved extracellular disulfide bridges and aromatic residues in extracellular loop 2 (ECL-2) for ligand binding and activation in the chemokine receptor CCR8. We used inositol 1,4,5-trisphosphate accumulation and radioligand binding experiments to determine the impact of receptor mutagenesis on both chemokine and small molecule agonist and antagonist binding and action in CCR8. We find that the seven-transmembrane (TM) receptor conserved disulfide bridge (7TM bridge) linking transmembrane helix III (TMIII) and ECL-2 is crucial for chemokine and small molecule action, whereas the chemokine receptor conserved disulfide bridge between the N terminus and TMVII is needed only for chemokines. Furthermore, we find that two distinct aromatic residues in ECL-2, Tyr184 (Cys + 1) and Tyr187 (Cys + 4), are crucial for binding of the CC chemokines CCL1 (agonist) and MC148 (antagonist), respectively, but not for small molecule binding. Finally, using in silico modeling, we predict an aromatic cluster of interaction partners for Tyr187 in TMIV (Phe171) and TMV (Trp194). We show in vitro that these residues are crucial for the binding and action of MC148, thus supporting their participation in an aromatic cluster with Tyr187. This aromatic cluster appears to be present in a large number of CC chemokine receptors and thereby could play a more general role to be exploited in future drug development targeting these receptors.

U2 - 10.1074/jbc.m115.706747

DO - 10.1074/jbc.m115.706747

M3 - Journal article

C2 - 27226537

SN - 0021-9258

VL - 291

SP - 16208

EP - 16220

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 31

ER -

Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding

Abstract

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