Identification of functionally important residues of human thrombopoietin.
Thrombopoietin (TPO) is a megakaryocyte growth and differentiation factor. It consists of a characteristic two domain structure. The amino-terminal domain of TPO has a sequence homology with erythropoietin and is required for the binding and activation of its receptor c-Mpl. To determine the functionally important regions interacting with its receptor, a series of site-directed mutants of TPO were constructed based on a three-dimensional model of the amino-terminal domain. Two strategies of mutagenesis were employed: 1) nonnative N-linked glycosylation scan of 12 residues predicted to be on the surface, and 2) alanine replacement scan of mostly charged 44 amino acid residues. Each TPO mutein was transiently expressed in COS7 cells, and the specific bioactivity of the TPO protein secreted into the culture medium was measured using a recombinant BaF3 cell line expressing human c-Mpl. Four alanine substitutions at Arg10, Pro42, Glu50, and Lys138 nearly or completely abolished the activity, whereas the mutation at Arg14 slightly decreased the activity, suggesting that these residues are functionally important in interacting with its receptor. These residues mapped to helix A, loop AB, and helix D. Sequence comparison between human TPO and other mammalian TPO showed that the identified residues are completely conserved among the species. However, unlike the recent report on the mutational analysis of TPO, alanine substitutions at Lys52, Lys59, Arg136, and Arg140 did not affect the TPO activity significantly in our system. The identified receptor binding regions of TPO are analogous to those of human growth hormone and erythropoietin. Based on the similarity of these three cytokines, we propose that Lys138 of helix D and Pro42 and Glu50 of loop AB may constitute one binding region, whereas Arg10 and Lys14 of helix A may constitute the other binding region to dimerize the receptors.
Park
H
H
Protein Engineering Research Group, Korea Research Institute of Bioscience and Biotechnology, KIST, P. O. Box 115, Yusong, Taejon 305-600, Korea.
Park
S S
SS
Jin
E H
EH
Song
J S
JS
Ryu
S E
SE
Yu
M H
MH
Hong
H J
HJ
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013926:Thrombopoietin / Q000737:chemistry* / Q000235:genetics
D018076:DNA, Complementary; D013926:Thrombopoietin; D000409:Alanine