Kinetic analysis of the early signaling steps of the human chemokine receptor CXCR4

Abstract

G protein–coupled receptors (GPCRs) are biologic switches that transduce extracellular stimuli into intracellular responses in the cell. Temporally resolving GPCR transduction pathways is key to understanding how cell signaling occurs. Here, we investigate the kinetics and dynamics of the activation and early signaling steps of the CXC chemokine receptor (CXCR) 4 in response to its natural ligands CXC chemokine ligand (CXCL) 12 and macrophage migration inhibitory factor (MIF), using Förster resonance energy transfer–based approaches. We show that CXCR4 presents a multifaceted response to CXCL12, with receptor activation (=0.6 seconds) followed by a rearrangement in the receptor/G protein complex (=1 seconds), a slower dimer rearrangement (=1.7 seconds), and prolonged G protein activation (=4 seconds). In comparison, MIF distinctly modulates every step of the transduction pathway, indicating distinct activation mechanisms and reflecting the different pharmacological properties of these two ligands. Our study also indicates that CXCR4 exhibits some degree of ligand-independent activity, a relevant feature for drug development.