Expression of a fusion protein of human ciliary neurotrophic factor and soluble CNTF-Receptor and identification of its activity

摘要

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 gpl30 and LIF-R, forming a tripartite receptor complex. Ceils that only express gpl30 and LIF-R, but not CNTF-R are refractory to stimulation by CNTF. On many target ceils 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 sac-cessfully 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 tmnsfected BAF/3 cells in response to CNTF and Hy-per-CNTF were used to verify the activity of the cytokines. The proliferative results confirmed that CNTF required homodimerization of the gpl30, CNTF-R and LIF-R receptor subunlt whereas Hyper-CNTF required heterodimerization of the gpl30 and LIF-R receptor subunit. We concluded that the fusion protein Hyper-CNTF had superagonistic activity on target cells expressing gpl30 and LIF-R, but lacking membrane-beund CNTF-R.