Received for publication June 14, 2004.
Revised October 26, 2004.
Accepted for publication October 26, 2004.

Evidence for negative binding cooperativity within CCR5-CCR2b heterodimers

Abstract

It is well established that most G protein-coupled receptors are able to form homo- and heterodimers, although the functional consequences of this process often remain unclear. CCR5 is a chemokine receptor that plays an important role in inflammatory diseases, and acts as a major co-receptor for human immunodeficiency viruses (HIV). CCR5 was previously shown to homodimerize and heterodimerize with CCR2b, a closely related receptor. In the present study, we have analyzed the functional consequences of this dimerization process, in terms of ligand binding, stimulation of intracellular cascades and internalization. BRET and co-immunoprecipitation assays demonstrated that CCR5 and CCR2b heterodimerize with the same efficiency as they homodimerize. In contrast to what has been reported earlier, no cooperative signaling was observed following co-stimulation of the two receptors by their respective ligands. However, we observed that CCR5-specific ligands which are unable to compete for MCP-1 binding on cells expressing CCR2b alone, efficiently prevented MCP-1 binding when CCR5 and CCR2b were co-expressed. The extent of this cross-competition was correlated with the amount of CCR5 expressed in cells, as determined by FACS analysis. Similar observations were made for the CCR2b-selective ligand MCP-1, that competed efficiently for MIP-1 binding on cells expressing both receptors. Internalization assays did not allow us to demonstrate co-internalization of the receptors in response to agonist stimulation. Taken together, our observations suggest that CCR5 and CCR2b form homo- and heterodimers with similar efficiencies, and that a receptor dimer can only bind a single chemokine.

Key words: Chemotactic peptides, Gi family, Calcium (G Protein Coupled Signals), Structure-activity relationships and modeling, Fluorescence techniques, Receptor binding studies