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

Abstract

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 125 I-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 nonpeptide 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.

Structural Motifs of Importance for the Constitutive Activity of the Orphan 7TM Receptor EBI2: Analysis of Receptor Activation in the Absence of an Agonist

“…Position V:12, which protrudes right into the major binding pocket, is conserved as a small amino acid (Gly in 29% and Leu in 14%), and in some receptors, introduction of larger side chains abolishes receptor activity (Jensen et al, 2008). Despite this, we observed only a minor decrease (1.6-fold) for the substitution of Gly for Trp in V:12 (Table 1).…”

AMD3100 Is a CXCR7 Ligand with Allosteric Agonist Properties

“…In this manner, a particular ligand-modulator-receptor complex may be capable of stabilizing a distinct conformation, and the effect of this modulation is unique to any given receptor and signaling pathway being studied, a phenomenon recently referred to as "functional selectivity" (Urban et al, 2007). The operational models illustrate that the effect of the allosteric ligand on the signaling output is entirely dependent on the probe with which it is interacting [i.e., probe dependence (Kenakin, 2005)], and this has been underscored recently with the same small-molecule allosteric agonist acting through the CCR1 receptor as either a positive or negative modulator depending upon the endogenous chemokine (probe) tested (Jensen et al, 2008). In the case of AMD3100, we have demonstrated that this ligand behaves differently against the same probe (CXCL12) at two different receptors (CXCR4 and CXCR7).…”

Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding

“…We used the metal ion chelator complexes bipyridine (Bip) and phenanthroline (Phe) in complex with zinc and copper (ZnBip, CuBip, ZnPhe, and CuPhe), which have been shown to activate CCR8 with micromolar potencies (31,33). These agonists bind to deeply located residues in the main binding pocket and are expected to be independent on extracellular receptor regions (34,35). The 7TM bridge was found to be important for small molecule-mediated activation, because alanine substitution of either Cys 106 or Cys 183 totally abolished activation, as exemplified by CuPhe (Fig.…”