title: Expression of a fusion protein of human ciliary neurotrophic factor and soluble CNTF-receptor and identification of its activity.
abstract: Ciliary neurotrophic factor (CNTF) has pleiotropic actions on many neuronal populations as well as on glia. Signal transduction by CNTF requires that it bind first to CNTF-R, permitting the recruitment of gp130 and LIF-R, forming a tripartite receptor complex. Cells that only express gp130 and LIF-R, but not CNTF-R are refractory to stimulation by CNTF. On many target cells CNTF only acts in the presence of its specific agonistic soluble receptors. We engineered a soluble fusion protein by linking the COOH-terminus of sCNTF-R to the NH2-terminus of CNTF. Recombinant CNTF/sCNTF-R fusion protein (Hyper-CNTF) was successfully expressed in COS-7 cells. The apparent molecular mass of the Hyper-CNTF protein was estimated from western blots to be 75 kDa. Proliferation assays of transfected BAF/3 cells in response to CNTF and Hyper-CNTF were used to verify the activity of the cytokines. The proliferative results confirmed that CNTF required homodimerization of the gp130, CNTF-R and LIF-R receptor subunit whereas Hyper-CNTF required heterodimerization of the gp130 and LIF-R receptor subunit. We concluded that the fusion protein Hyper-CNTF had superagonistic activity on target cells expressing gp130 and LIF-R, but lacking membrane-bound CNTF-R.
authors.affiliation: College of Life Sciences, Zhejiang University, Hangzhou 310027, China. sun_yi@mail.hz.zj.cn
authors.forename: Yi
authors.initials: Y
authors.lastname: Sun
authors.forename: März
authors.initials: M
authors.lastname: Pia
authors.forename: Otten
authors.initials: O
authors.lastname: Uwe
authors.forename: Ji-Guang
authors.initials: JG
authors.lastname: Ge
authors.forename: Rose-John
authors.initials: RJ
authors.lastname: Stefan
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D019556:COS Cells / Q000166:cytology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002522:Chlorocebus aethiops; D020934:Ciliary Neurotrophic Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D050822:Cytokine Receptor gp130; D016207:Cytokines / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008970:Molecular Weight; D015202:Protein Engineering / Q000379:methods; D020801:Receptor, Ciliary Neurotrophic Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018121:Receptors, Cytokine / Q000378:metabolism*; D053606:Receptors, OSM-LIF; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D012995:Solubility
chemical_list: D015703:Antigens, CD; D020934:Ciliary Neurotrophic Factor; D016207:Cytokines; C494199:IL6ST protein, human; C507840:LIFR protein, human; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit; D008562:Membrane Glycoproteins; D020801:Receptor, Ciliary Neurotrophic Factor; D018121:Receptors, Cytokine; D053606:Receptors, OSM-LIF; D011993:Recombinant Fusion Proteins; D050822:Cytokine Receptor gp130