title: Receptor binding and cellular uptake studies of macrophage migration inhibitory factor (MIF): use of biologically active labeled MIF derivatives.
abstract: Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine for which a receptor has not been identified. That MIF has intracellular functions has been suggested by its enzymatic activity and constitutive expression profile. The discovery of functional MIF-c-Jun activation domain binding protein 1 (JAB1) binding has confirmed this notion and indicated that nonreceptor-based signaling mechanisms are important for MIF function. Here, we have generated and tested several biologically active labeled MIF derivatives to further define target protein binding by MIF and its cellular uptake characteristics. (35)S-MIF, biotinylated MIF, and fluoresceinated MIF were demonstrated to exhibit full biologic activity. Neither by applying a standard iodinated MIF preparation nor by using the biologically active (35)S-MIF derivative in receptor-binding studies were we able to measure any receptor-binding activity on numerous cells, confirming that uptake of MIF into target cells and MIF signaling can occur by receptor-independent pathways. When MIF derivatives were applied in cellular uptake studies, MIF was found to be endocytosed into both immune and nonimmune cells and targeted to the cytosol and lysosomes. The entry of MIF was temperature and energy dependent and was inhibited by monodansylcadaverine but not by ouabain. Endocytosed biotin-MIF bound JAB1 not only in macrophages, as shown previously, but also in nonimmune cells. A tagged MIF construct, MIF-enhanced green fluorescent protein (EGFP), was shown to be a valuable tool, as EGFP constructs of critical MIF cysteine mutants exhibited identical cellular localization properties to those of wild-type MIF (wtMIF). Our results indicate that MIF membrane receptors are not widely expressed, if at all, and suggest that the cellular uptake of MIF occurs by nonreceptor-mediated endocytosis rather than penetration. All the derivatives investigated, except for iodinated MIF, represent valuable tools for further MIF target protein and cellular studies.
authors.affiliation: Laboratory of Biochemistry, Institute for Interfacial Engineering, University of Stuttgart, D-70569 Stuttgart, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Kleemann
authors.forename: Matthias
authors.initials: M
authors.lastname: Grell
authors.forename: Ralf
authors.initials: R
authors.lastname: Mischke
authors.forename: Gudrun
authors.initials: G
authors.lastname: Zimmermann
authors.forename: Jürgen
authors.initials: J
authors.lastname: Bernhagen
mesh_terms: D016475:3T3 Cells; D000818:Animals; D019921:Biotinylation; D000075686:COP9 Signalosome Complex; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004705:Endocytosis; D005452:Fluoresceins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D020014:K562 Cells; D007739:L Cells; D008247:Lysosomes / Q000378:metabolism; D008263:Macrophage Migration-Inhibitory Factors / Q000378:metabolism* / Q000502:physiology; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D010447:Peptide Hydrolases; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000502:physiology; D013194:Staining and Labeling; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D005452:Fluoresceins; D047908:Intracellular Signaling Peptides and Proteins; D008263:Macrophage Migration-Inhibitory Factors; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D010447:Peptide Hydrolases; C501488:COPS5 protein, human; C501490:Cops5 protein, mouse; D000075686:COP9 Signalosome Complex