TY - JOUR

T1 - Extracellular Disulfide Bridges Serve Different Purposes in Two Homologous Chemokine Receptors, CCR1 and CCR5

AU - Rummel, Pia Cwarzko

AU - Thiele, Stefanie

AU - Hansen, Laerke Smidt

AU - Petersen, Trine Puggaard

AU - Sparre-Ulrich, Alexander Hovard

AU - Ulven, Trond

AU - Rosenkilde, Mette Marie

PY - 2013/6/13

Y1 - 2013/6/13

N2 - In addition to the 7TM receptor-conserved disulfide bridge between transmembrane helix (TM) 3 and extracellular loop (ECL) 2, chemokine receptors contain a disulfide bridge between the N-terminus and what previously was believed to be ECL-3. Recent crystal- and NMR-structures of CXCR4 and CXCR1, combined with structural analysis of all endogenous chemokine receptors indicate that this chemokine receptor-conserved bridge in fact connects the N-terminus to the top of TM-7. By employing chemokine ligands that mainly target extracellular receptor regions and small molecule ligands that predominantly interact with residues in the main binding crevice, we show that the 7TM-conserved bridge is essential for all types of ligand-mediated activation, whereas the chemokine-conserved bridge is dispensable for small-molecule activation in CCR1. However, in striking contrast to previous studies in other chemokine receptors, high affinity CCL3 chemokine binding was maintained in the absence of either bridge. In CCR5, the closest homolog to CCR1, a completely different dependency was observed as neither chemokine activation nor binding was retained in the absence of either bridge. In contrast, both bridges where dispensable for small-molecule activation. This indicates that CCR5 activity is independent of extracellular regions, whereas in CCR1, preserved folding of ECL2 is necessary for activation. These results indicate that conserved structural features in a receptor subgroup, does not necessarily provide specific traits for the whole subgroup, but rather provides unique traits to the single receptors.

AB - In addition to the 7TM receptor-conserved disulfide bridge between transmembrane helix (TM) 3 and extracellular loop (ECL) 2, chemokine receptors contain a disulfide bridge between the N-terminus and what previously was believed to be ECL-3. Recent crystal- and NMR-structures of CXCR4 and CXCR1, combined with structural analysis of all endogenous chemokine receptors indicate that this chemokine receptor-conserved bridge in fact connects the N-terminus to the top of TM-7. By employing chemokine ligands that mainly target extracellular receptor regions and small molecule ligands that predominantly interact with residues in the main binding crevice, we show that the 7TM-conserved bridge is essential for all types of ligand-mediated activation, whereas the chemokine-conserved bridge is dispensable for small-molecule activation in CCR1. However, in striking contrast to previous studies in other chemokine receptors, high affinity CCL3 chemokine binding was maintained in the absence of either bridge. In CCR5, the closest homolog to CCR1, a completely different dependency was observed as neither chemokine activation nor binding was retained in the absence of either bridge. In contrast, both bridges where dispensable for small-molecule activation. This indicates that CCR5 activity is independent of extracellular regions, whereas in CCR1, preserved folding of ECL2 is necessary for activation. These results indicate that conserved structural features in a receptor subgroup, does not necessarily provide specific traits for the whole subgroup, but rather provides unique traits to the single receptors.

U2 - 10.1124/mol.113.086702

DO - 10.1124/mol.113.086702

M3 - Journal article

C2 - 23765404

VL - 84

SP - 335

EP - 345

JO - Current Molecular Pharmacology

JF - Current Molecular Pharmacology

SN - 1874-4702

IS - 3

ER -