Extracellular disulfide bridges serve different purposes in two homologous chemokine receptors, CCR1 and CCR5.

In addition to the 7 transmembrane receptor (7TM)-conserved disulfide bridge between transmembrane (TM) helix 3 and extracellular loop (ECL)-2, chemokine receptors (CCR) contain a disulfide bridge between the N terminus and what previously was believed to be ECL-3. Recent crystal and NMR structures of the CXC chemokine receptors (CXCR) 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 the highly related CCR5, a completely different dependency was observed as neither activation nor binding of the same chemokines was retained in the absence of either bridge. In contrast, both bridges were dispensable for activation by the same small molecules. This indicates that CCR5 activity is independent of extracellular regions, whereas in CCR1 the preserved folding of ECL-2 is necessary for activation. These results indicate that conserved structural features in a receptor subgroup do not necessarily provide specific traits for the whole subgroup but rather provide unique traits to the single receptors.

Biased and constitutive signaling in the CC-chemokine receptor CCR5 by manipulating the interface between transmembrane helices 6 and 7.

The equilibrium state of CCR5 is manipulated here toward either activation or inactivation by introduction of single amino acid substitutions in the transmembrane domains (TMs) 6 and 7. Insertion of a steric hindrance mutation in the center of TM7 (G286F in position VII:09/7.42) resulted in biased signaling. Thus, ß-arrestin recruitment was eliminated, whereas constitutive activity was observed in Gαi-mediated signaling. Furthermore, the CCR5 antagonist aplaviroc was converted to a full agonist (a so-called efficacy switch). Computational modeling revealed that the position of the 7TM receptor-conserved Trp in TM6 (Trp-248 in position VI:13/6.48, part of the CWXP motif) was influenced by the G286F mutation, causing Trp-248 to change orientation away from TM7. The essential role of Trp-248 in CCR5 activation was supported by complete inactivity of W248A-CCR5 despite maintaining chemokine binding. Furthermore, replacing Trp-248 with a smaller aromatic amino acid (Tyr/Phe) impaired the ß-arrestin recruitment, yet with maintained G protein activity (biased signaling); also, here aplaviroc switched to a full agonist. Thus, the altered positioning of Trp-248, induced by G286F, led to a constraint of G protein active, but ß-arrestin inactive and thus biased, CCR5 conformation. These results provide important information on the molecular interplay and impact of TM6 and TM7 for CCR5 activity, which may be extrapolated to other chemokine receptors and possibly to other 7TM receptors.