Evaluation and extension of the two-site, two-step model for binding and activation of the chemokine receptor CCR1

Julie Sanchez,Zil E Huma,J Robert Lane,Xuyu Liu,Jessica L Bridgford,Richard J Payne,Meritxell Canals,Martin J Stone

doi:10.1074/jbc.ra118.006535

Abstract

Interactions between secreted immune proteins called chemokines and their cognate G protein-coupled receptors regulate the trafficking of leukocytes in inflammatory responses. The two-site, two-step model describes these interactions. It involves initial binding of the chemokine N-loop/β3 region to the receptor's N-terminal region and subsequent insertion of the chemokine N-terminal region into the transmembrane helical bundle of the receptor concurrent with receptor activation. Here, we test aspects of this model with C-C motif chemokine receptor 1 (CCR1) and several chemokine ligands. First, we compared the chemokine-binding affinities of CCR1 with those of peptides corresponding to the CCR1 N-terminal region. Relatively low affinities of the peptides and poor correlations between CCR1 and peptide affinities indicated that other regions of the receptor may contribute to binding affinity. Second, we evaluated the contributions of the two CCR1-interacting regions of the cognate chemokine ligand CCL7 (formerly monocyte chemoattractant protein-3 (MCP-3)) using chimeras between CCL7 and the non-cognate ligand CCL2 (formerly MCP-1). The results revealed that the chemokine N-terminal region contributes significantly to binding affinity but that differences in binding affinity do not completely account for differences in receptor activation. On the basis of these observations, we propose an elaboration of the two-site, two-step model-the "three-step" model-in which initial interactions of the first site result in low-affinity, nonspecific binding; rate-limiting engagement of the second site enables high-affinity, specific binding; and subsequent conformational rearrangement gives rise to receptor activation.

Highlights

Interactions between secreted immune proteins called chemokines and their cognate G protein– coupled receptors regulate the trafficking of leukocytes in inflammatory responses
If the site 1 interactions make a major contribution to the binding interactions, one would anticipate that the binding free energy of such N-terminal peptides for chemokines would be a substantial proportion of the binding free energy of intact receptors for the same chemokines
It would be expected that the binding free energies of several chemokines would correlate for the N-terminal peptides and the intact receptors

Summary

Introduction

Interactions between secreted immune proteins called chemokines and their cognate G protein– coupled receptors regulate the trafficking of leukocytes in inflammatory responses. The two-site, two-step model describes these interactions It involves initial binding of the chemokine N-loop/␤3 region to the receptor’s N-terminal region and subsequent insertion of the chemokine N-terminal region into the transmembrane helical bundle of the receptor concurrent with receptor activation. Chemokines bind to and activate chemokine receptors, which are G protein– coupled receptors (GPCRs) expressed in leukocyte cell membranes. This initiates intracellular signal transduction, leading to changes in leukocyte morphology and adhesion and giving rise to accumulation of leukocytes in the affected tissues. The chemokine “N-loop” and nearby ␤3 region, together defined as chemokine site 1 (CS1), were found to bind to peptides corresponding to the flexible N-terminal regions of chemokine receptors, defined

Results

Discussion

Conclusion