Identification of full, partial and inverse CC chemokine receptor 3 agonists using [35S]GTPγS binding

Abstract Study of the CC chemokine receptor 3 (CCR3) has been limited to using radiolabeled agonist chemokines. A small molecule CCR3 antagonist, 2-[(6-amino-2-benzothiazolyl)thio]- N -[1-[(3,4-dichlorylphenyl)methyl]-4-piperidinyl]acetamide, Banyu (I), was tritiated and used for pharmacological studies. Banyu (I) has a K d of 5.0±0.4 and 4.3±1.8 nM on human CCR3 transfectants and eosinophils, and noncompetitively inhibits [ 125 I]eotaxin binding and eotaxin-induced [ 35 S]guanosine-5′- O -(3-thiotriphosphate) ([ 35 S]GTPγS) binding. The proportion of [ 125 I]eotaxin: [ 3 H]Banyu (I) binding sites in eosinophils or transfectants was 35% or 13%, although both binding sites were overexpressed in transfectants. CCR3 spontaneously couples to G-proteins in CCR3 transfectants, demonstrated by changes in basal and eotaxin-induced [ 35 S]GTPγS binding under reduced NaCl and GDP concentrations. Consequently, Banyu (I) was identified as an inverse agonist. In contrast, CCL18 and I-TAC (interferon-inducible T cell α-chemoattractant) were neutral antagonists, inhibiting eotaxin-induced [ 35 S]GTPγS binding, with minimal effect on basal coupling of CCR3 to G proteins. Eotaxin, eotaxin-2 and monocyte chemoattractant protein (MCP)-4 are full agonists inducing [ 35 S]GTPγS binding; eotaxin-3, MCP-3, RANTES (regulated on activation normal T cell expressed and secreted), vMIP-I (Kaposi's sarcoma-associated herpesvirus macrophage inflammatory protein-) and vMIP-II are partial agonists, indicating that this is a sensitive method to quantitate agonist efficacy.