title: Chemokine receptor-ligand interactions measured using time-resolved fluorescence.
abstract: Two G protein-coupled receptor subtypes (CXCR1 and CXCR2) mediate Interleukin-8 (IL8) action in cells. A nonradioactive lanthanide-chelate derivatized IL8 ligand was developed to measure the binding activity of the chemokine receptors, CXCR1 and CXCR2. Site-specific mutagenesis of the carboxyl-terminal serine of IL8 to cysteine resulted in a mutant IL8 (IL8-S72C) having a single free sulfhydryl. Using an iodoacetamide derivative of the Eu3+-chelate of N-(p-benzoic acid)diethylenetriamine-N,N',N"-tetraacetic acid (DTTA), incorporation of one Eu3+ per IL8 molecule ([Eu3+]IL8-S72C) was achieved. The dissociation constant for this conjugate was similar to that measured for [125I]IL8 ( approximately 2 nM) when measured by time-resolved fluorometry using CHO cell lines stably expressing CXCR1 or CXCR2 receptors. The sensitivity, stability, and high specific activity of europium-labeled IL8 demonstrate the usefulness of lanthanide-labeled proteins in the measurement of receptor-ligand interactions and may be extended to other peptide ligands.
authors.affiliation: Pharmacopeia Inc., 101 College Road East, Princeton, New Jersey 08540, USA. inglese@pharmacop.com
authors.forename: J
authors.initials: J
authors.lastname: Inglese
authors.forename: P
authors.initials: P
authors.lastname: Samama
authors.forename: S
authors.initials: S
authors.lastname: Patel
authors.forename: J
authors.initials: J
authors.lastname: Burbaum
authors.forename: I L
authors.initials: IL
authors.lastname: Stroke
authors.forename: K C
authors.initials: KC
authors.lastname: Appell
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D001896:Boron Compounds; D016466:CHO Cells; D002614:Chelating Agents; D006224:Cricetinae; D005063:Europium; D019793:Fluorescein; D005456:Fluorescent Dyes; D006801:Humans; D016209:Interleukin-8 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D019707:Receptors, Chemokine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018123:Receptors, Interleukin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D023062:Receptors, Interleukin-8A; D023063:Receptors, Interleukin-8B; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012235:Rhodamines; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D015703:Antigens, CD; D001896:Boron Compounds; D002614:Chelating Agents; D005456:Fluorescent Dyes; D016209:Interleukin-8; D008024:Ligands; D019707:Receptors, Chemokine; D018123:Receptors, Interleukin; D023062:Receptors, Interleukin-8A; D023063:Receptors, Interleukin-8B; D011994:Recombinant Proteins; D012235:Rhodamines; D005063:Europium; D019793:Fluorescein