Received for publication September 30, 2005.
Revised February 7, 2006.
Accepted for publication February 7, 2006.

Allosteric modulation of binding properties between units of chemokine receptor homo- and hetero-oligomers

Abstract

We have previously demonstrated that the chemokine receptors CCR2 and CCR5 form homo- and heterodimers, and that dimers can only bind a single chemokine molecule with high affinity. We provide here evidence from BRET experiments that stimulation by chemokines does not influence the CCR2/CCR5 heterodimerization status. In addition, we show that the rate of radioligand dissociation from one unit of the heterodimer in "infinite" tracer dilution conditions is strongly increased in the presence of an unlabelled chemokine ligand of the other unit. These results demonstrate unambiguously that the interaction between heterodimer units is of allosteric nature. Agonists, but also some monoclonal antibodies, could promote such negative binding cooperativity, indicating that this phenomenom does not require the full conformational change associated with receptor activation. Finally, we show that G protein coupling is required for high affinity binding of MIP-1 (CCL4) to CCR5, and that the dissociation from G proteins, following incubation with Gpp(NH)p, promotes the release of pre-bound radiolabelled chemokines with kinetics similar to that measured after the addition of an excess of unlabelled chemokines. These observations suggest that the association with G proteins likely participates in the negative cooperativity observed between receptor monomers. We propose that negative cooperativity within homo- and hetero-dimers of chemokine receptors and probably other GPCRs will likely have major implications in their pharmacology in vivo and the physiopathology of diseases with which they are associated.

Key words: Chemotactic peptides, Gi family, Calcium (G Protein Coupled Signals), Desensitization/uncoupling, Fluorescence techniques, Receptor binding studies

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