Chemokine receptor-ligand interactions measured using time-resolved fluorescence

被引：48

作者：

Inglese, J ^{[1
]}

Samama, P ^{[1
]}

Patel, S ^{[1
]}

Burbaum, J ^{[1
]}

Stroke, IL ^{[1
]}

Appell, KC ^{[1
]}

机构：

[1] Pharmacopeia Inc, Princeton, NJ 08540 USA

来源：

BIOCHEMISTRY | 1998年 / 37卷 / 08期

关键词：

D O I：

10.1021/bi972161u

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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 [I-125]IL8 (similar to 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.

引用

页码：2372 / 2377

页数：6

共 31 条

[1] The CXC chemokines growth-regulated oncogene (GRO) alpha, GRO beta, GRO gamma, neutrophil-activating peptide-2, and epithelial cell-derived neutrophil-activating peptide-78 are potent agonists for the type B, but not the type A, human interleukin-8 receptor [J].