title: Molecular proximity of complement receptor type 3 (CR3) and the glycosylphosphatidylinositol-linked protein GPI-80 on neutrophils: effects of cell adherence, exogenous saccharides, and lipid raft disrupting agents.
abstract: GPI-80, a novel glycosylphosphatidylinositol (GPI)-anchored protein on polymorphonuclear leukocytes, has been reported to cooperate with CR3 in several aspects of cell function including cell activation, adhesion and migration. The present study investigates the physical proximity of CR3 and GPI-80 on living cells using resonance energy transfer (RET) techniques, which gives positive results when the separation distance is < or = 7 nm. RET from donor-labeled anti-CR3 to acceptor-labeled anti-GPI-80 was detected on adherent neutrophils, but not observed for non-adherent cells. Furthermore, RET was not observed on cells treated with cell adhesion inhibitors 4-bromophenacyl bromide (BPB), N-ethylmaleimide (NEM) or cytochalasin D, suggesting dynamic interactions between CR3 and GPI-80. CR3-to-GPI-80 proximity was blocked by N-acetyl-D-glucosamine (NADG), but not by other monosaccharides such as D-mannose, fructose, fucose, glucose, sorbitol, or galactose; molecular proximity was also disrupted by the glycolipid raft depleting agents 2-OH-propyl-betaCD and MbetaCD. Thus, lipid rafts may be important for the physical and functional cooperation of CR3 and GPI-80.
authors.affiliation: Department of Ophthalmology and Visual Science, University of Michigan Medical School, 1000 Wall Street, Ann Arbor, MI 48105, USA.
authors.forename: Ji-Biao
authors.initials: JB
authors.lastname: Huang
authors.forename: Yuji
authors.initials: Y
authors.lastname: Takeda
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Araki
authors.forename: Fujiro
authors.initials: F
authors.lastname: Sendo
authors.forename: Howard R
authors.initials: HR
authors.lastname: Petty
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism; D000581:Amidohydrolases; D000818:Animals; D002448:Cell Adhesion / Q000502:physiology; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000378:metabolism*; D058851:GPI-Linked Proteins; D006801:Humans; D006867:Hydrolases; D051379:Mice; D009504:Neutrophils / Q000378:metabolism*; D011951:Receptors, Complement / Q000737:chemistry / Q000378:metabolism*; D013057:Spectrum Analysis
chemical_list: D015815:Cell Adhesion Molecules; D058851:GPI-Linked Proteins; D011951:Receptors, Complement; D006867:Hydrolases; D000581:Amidohydrolases; C121771:VNN2 protein, human; D000117:Acetylglucosamine