Signal transmission through the CXC chemokine receptor 4 (CXCR4) transmembrane helices

Abstract

The atomic-level mechanisms by which G protein-coupled receptors (GPCRs) transmit extracellular ligand binding events through their transmembrane helices to activate intracellular G proteins remain unclear. Using a comprehensive library of mutations covering all 352 residues of the GPCR CXC chemokine receptor 4 (CXCR4), we identified 41 amino acids that are required for signaling induced by the chemokine ligand CXCL12 (stromal cell-derived factor 1). CXCR4 variants with each of these mutations do not signal properly but remain folded, based on receptor surface trafficking, reactivity to conformationally sensitive monoclonal antibodies, and ligand binding. When visualized on the structure of CXCR4, the majority of these residues form a continuous intramolecular signaling chain through the transmembrane helices; this chain connects chemokine binding residues on the extracellular side of CXCR4 to G proteincoupling residues on its intracellular side. Integrated into a cohesive model of signal transmission, these CXCR4 residues cluster into five functional groups that mediate (i) chemokine engagement, (ii) signal initiation, (iii) signal propagation, (iv) microswitch activation, and (v) G protein coupling. Propagation of the signal passes through a "hydrophobic bridge" on helix VI that coordinates with nearly every known GPCR signaling motif. Our results agree with known conserved mechanisms of GPCR activation and significantly expand on understanding the structural principles of CXCR4 signaling.T he CXC chemokine receptor 4 (CXCR4) belongs to the G protein-coupled receptor (GPCR) superfamily of proteins, the largest class of integral membrane proteins encoded in the human genome, comprising greater than 30% of current drug targets. Deregulation of CXCR4 expression in multiple human cancers, its role in hematopoietic stem cell migration, and the utilization of CXCR4 by HIV-1 for T-cell entry, make this receptor an increasingly important therapeutic target (1). One FDA-approved drug against CXCR4 is currently on the market (Mozobil, for hematopoietic stem cell mobilization), and multiple additional drugs against this target are in development for oncology and other indications (2).The crystal structures of class A GPCR superfamily members in their active and inactive conformations (reviewed in refs. 3 and 4) provide unprecedented insight into the structural basis of ligand binding, G protein coupling, and activation of GPCRs via rearrangements of transmembrane (TM) helices. GPCR helices V and VI in particular, and in some cases III and VII, are known to undergo significant conformational changes upon activation (5-7). However, static images alone have not been able to explain the residue-level mechanisms underlying the dynamic helical shifts that mediate GPCR signal transduction. Additionally, only inactive state structures have been solved for CXCR4 and most other GPCRs (8,9). Over the last two decades, extensive mutagenesis studies of GPCRs in general [collectively describing >8,000 mutations (gpcrdb.org)] and of CX...

What Do Structures Tell Us About Chemokine Receptor Function and Antagonism?

“…In addition to CRS1/1.5 interactions involving the N-terminus of the receptor proximal to the first conserved Cys, several reports suggest involvement of the distal N-terminus. Mutation or deletion of amino acids within the first 10 residues of the receptor have been shown to affect chemokine binding and/or receptor activation in the case of CXCR4:CXCL12 (12, 141) and CXCR2 with CXCL1, CXCL7 and CXCL8 (62). An NMR structure of CXCL12 in complex with an N-terminal peptide (residues 1–38) from CXCR4 suggests that the distal end pairs with the β 1 -strand of CXCL12 (119), a region also implicated by cross-saturation NMR experiments (67).…”

“…The extent and distributed nature of receptor:chemokine interfaces explain why mutation of individual receptor or chemokine residues, particularly involving CRS1, rarely has a dramatic impact on binding or signaling (141). Unless the mutated residue is an interaction hotspot (9), multiple mutations or truncations are typically required to produce a significant effect.…”

“…However, these static structures do not reveal the intricate and dynamic details of the residue network that couples agonist binding at the extracellular side to the intracellular conformational changes. Recent mutagenesis and radiolytic footprinting efforts, in combination with structural models, provided important residue-specific clues to the mechanism in the context of CXCR4 and ACKR3, respectively (48, 141). …”

“…Using high throughput shotgun mutagenesis covering every amino acid of CXCR4, Wescott and coworkers identified residue positions where mutations disrupted CXCL12-induced receptor signaling (141). The mutations may exert their effect through several mechanisms such as biasing the receptor conformational equilibrium, disrupting chemokine or G protein binding, or disrupting the intramolecular communication network.…”

“…These residues were clustered into five functional layers and their roles tentatively assigned as (i) chemokine engagement, (ii) signal initiation, (iii) signal propagation, (iv) activation microswitches and (v) G protein binding (Fig. 3a–f) (141). …”