title: The human CC chemokine MIP-1beta dimer is not competent to bind to the CCR5 receptor.
abstract: Chemokine dimerization has been the subject of much interest in recent years as evidence has accumulated that different quaternary states of chemokines play different biological roles; the monomer is believed to be the receptor-binding unit, whereas the dimer has been implicated in binding cell surface glycosaminoglycans. However, although several studies have provided evidence for this paradigm by making monomeric chemokine variants or dimer-impaired chemokines, few have provided direct evidence of the receptor function of a chemokine dimer. We have produced a covalent dimer of the CC chemokine macrophage inflammatory protein-1beta (MIP-1beta) by placing a disulfide bond at the center of its dimer interface through a single amino acid substitution (MIP-1beta-A10C). This variant was shown to be a nondissociating dimer by SDS-PAGE and analytical ultracentrifugation. NMR reveals a structure largely the same as the wild type protein. In studies of glycosaminoglycan binding, MIP-1beta-A10C binds to a heparin-Sepharose column as tightly as the wild type protein and more tightly than monomeric variants. However, MIP-1beta-A10C neither binds nor activates the MIP-1beta receptor CCR5. It was found that the ability to activate CCR5 was recovered upon reduction of the intermolecular disulfide cross-link by incubation with 1 mm dithiothreitol. This work provides the first definitive evidence that the CC chemokine MIP-1beta dimer is not able to bind or activate its receptor and implicates the CC chemokine monomer as the sole receptor-interacting unit.
authors.affiliation: Department of Biochemistry and Biophysics, Texas A & M University, College Station, Texas 77843, USA.
authors.forename: Hongjun
authors.initials: H
authors.lastname: Jin
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Shen
authors.forename: Brandi Renee
authors.initials: BR
authors.lastname: Baggett
authors.forename: Xiangming
authors.initials: X
authors.lastname: Kong
authors.forename: Patricia J
authors.initials: PJ
authors.lastname: LiWang
mesh_terms: D000818:Animals; D016466:CHO Cells; D054407:Chemokine CCL4; D006224:Cricetinae; D003412:Cricetulus; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019281:Dimerization; D004229:Dithiothreitol / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D019402:Macrophage Inflammatory Proteins / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D011485:Protein Binding; D019713:Receptors, CCR5 / Q000737:chemistry* / Q000378:metabolism; D012685:Sepharose / Q000737:chemistry
chemical_list: D054407:Chemokine CCL4; D003432:Cross-Linking Reagents; D019402:Macrophage Inflammatory Proteins; D019713:Receptors, CCR5; D012685:Sepharose; D004229:Dithiothreitol