title: Identification of full, partial and inverse CC chemokine receptor 3 agonists using [35S]GTPgammaS 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 [125I]eotaxin binding and eotaxin-induced [35S]guanosine-5'-O-(3-thiotriphosphate) ([35S]GTPgammaS) binding. The proportion of [125I]eotaxin: [3H]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 [35S]GTPgammaS 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 alpha-chemoattractant) were neutral antagonists, inhibiting eotaxin-induced [35S]GTPgammaS 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 [35S]GTPgammaS 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.
authors.affiliation: Department of Allergy, Schering-Plough Research Institute, Kenilworth, NJ 07033, USA.
authors.forename: Yuntao
authors.initials: Y
authors.lastname: Wan
authors.forename: James P
authors.initials: JP
authors.lastname: Jakway
authors.forename: Hongchen
authors.initials: H
authors.lastname: Qiu
authors.forename: Himanshu
authors.initials: H
authors.lastname: Shah
authors.forename: Charles G
authors.initials: CG
authors.lastname: Garlisi
authors.forename: Fang
authors.initials: F
authors.lastname: Tian
authors.forename: Pauline
authors.initials: P
authors.lastname: Ting
authors.forename: David
authors.initials: D
authors.lastname: Hesk
authors.forename: R W
authors.initials: RW
authors.lastname: Egan
authors.forename: M Motasim
authors.initials: MM
authors.lastname: Billah
authors.forename: Shelby P
authors.initials: SP
authors.lastname: Umland
mesh_terms: D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D054413:Chemokine CCL11; D018946:Chemokine CCL5 / Q000378:metabolism / Q000494:pharmacology; D018925:Chemokines / Q000378:metabolism / Q000494:pharmacology; D019742:Chemokines, CC / Q000378:metabolism / Q000494:pharmacology; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004804:Eosinophils / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006801:Humans; D018945:Monocyte Chemoattractant Proteins / Q000378:metabolism / Q000494:pharmacology; D011869:Radioligand Assay; D051381:Rats; D054397:Receptors, CCR3; D019707:Receptors, Chemokine / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014162:Transfection
chemical_list: C515456:CCL11 protein, human; C099677:CCL13 protein, human; C515350:CCR3 protein, human; C515458:Ccl11 protein, rat; C515352:Ccr3 protein, rat; D054413:Chemokine CCL11; D018946:Chemokine CCL5; D018925:Chemokines; D019742:Chemokines, CC; D018945:Monocyte Chemoattractant Proteins; D054397:Receptors, CCR3; D019707:Receptors, Chemokine; D013462:Sulfur Radioisotopes; D016244:Guanosine 5'-O-(3-Thiotriphosphate)