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J. Biol. Chem., Vol. 283, Issue 34, 23121-23128, August 22, 2008

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Positive Versus Negative Modulation of Different Endogenous Chemokines for CC-chemokine Receptor 1 by Small Molecule Agonists through Allosteric Versus Orthosteric Binding^*

Pia C. Jensen¹, Stefanie Thiele¹, Trond Ulven, Thue W. Schwartz, and Mette M. Rosenkilde²

From the Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Copenhagen University, Blegdamsvej 3, DK-2200, Copenhagen, Denmark and the Department of Physics and Chemistry, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark

7 transmembrane-spanning (7TM) chemokine receptors having multiple endogenous ligands offer special opportunities to understand the molecular basis for allosteric mechanisms. Thus, CC-chemokine receptor 1 (CCR1) binds CC-chemokine 3 and 5 (CCL3 and CCL5) with K_d values of 7.3 and 0.16 nM, respectively, as determined in homologous competition binding assays. However, CCL5 appears to have a >10,000-fold lower affinity in competition against ¹²⁵I-CCL3. Mutational mapping revealed that CCL3 and CCL5 both are strongly affected by systematic truncations of the N-terminal extension, whereas only CCL5 and not CCL3 activation is affected by substitutions in the main ligand binding pocket including the conserved GluVII:06 anchor point. A series of metal ion chelator complexes were found to act as full agonists on CCR1 and to be critically affected by the same substitutions in the main ligand binding pocket as CCL5 but not by mutations in the extracellular domain. In agreement with the overlapping binding sites, the small non-peptide agonists displaced radiolabeled CCL5 with high affinity. Interestingly, the same compounds acted as allosteric enhancers of the binding of CCL3, with which they did not overlap in binding site, leading to an increased B_max and affinity of this chemokine mainly due to an increased association rate. It is concluded that a small molecule agonist through binding deep in the main ligand binding pocket can act as an allosteric enhancer for one endogenous chemokine and at the same time as a competitive blocker of the binding of another endogenous chemokine.

Received for publication, May 6, 2008 , and in revised form, June 9, 2008.

^* This work was supported by the Danish Medical Research Council, the European Community's Sixth Framework Program (INNOCHEM: LSHB-CT-2005-518167), the NovoNordisk Foundation, the AP-Moller foundation, and the Aase og Einer Danielsen Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Laboratory for Molecular Pharmacology, Dept. of Neuroscience and Pharmacology, The Panum Institute, Copenhagen University, Blegdamsvej 3, DK-2200, Copenhagen, Denmark. Tel.: 0045-61364871; Fax: 0045 35327610; E-mail: rosenkilde{at}sund.ku.dk.

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