Identification of Functionally Important Residues of Human Thrombopoietin

摘要: Thrombopoietin (TPO) is a megakaryocyte growth and differentiation factor. It consists of characteristic two domain structure. The amino-terminal TPO has sequence homology with erythropoietin required for the binding activation its receptor c-Mpl. To determine functionally important regions interacting receptor, series site-directed mutants were constructed based on three-dimensional model domain. Two strategies mutagenesis employed: 1) nonnative N-linked glycosylation scan 12 residues predicted to be surface, 2) alanine replacement mostly charged 44 amino acid residues. Each mutein was transiently expressed in COS7 cells, specific bioactivity protein secreted into culture medium measured using recombinant BaF3 cell line expressing human Four substitutions at Arg10, Pro42, Glu50, Lys138 nearly or completely abolished activity, whereas mutation Arg14 slightly decreased suggesting that these are receptor. These mapped helix A, loop AB, D. Sequence comparison between other mammalian showed identified conserved among species. However, unlike recent report mutational analysis TPO, Lys52, Lys59, Arg136, Arg140 did not affect activity significantly our system. analogous those hormone erythropoietin. Based similarity three cytokines, we propose D Pro42 Glu50 AB may constitute one region, Arg10 Lys14 A region dimerize receptors.