title: Mutational analysis of thrombopoietin for identification of receptor and neutralizing antibody sites.
abstract: Thrombopoietin (TPO) is a hematopoietin important for megakaryocyte proliferation and production of blood platelets. We sought to characterize how TPO binds and activates its receptor, myeloproliferative leukemia virus receptor. The erythropoietin-like domain of TPO (TPO1-153) has been fused to the gIII coat protein of M13 bacteriophage. Forty residues were chosen for mutation to alanine using the criteria that they were charged residues or predicted to be solvent-exposed, based on a homology model. Phage enzyme-linked immunosorbent assay was used to determine affinities for binding to both the TPO receptor and five anti-TPO1-153 monoclonal antibodies. Mutations at mostly positively charged residues (Asp8, Lys14, Lys52, Lys59, Lys136, Lys138, Arg140) caused the greatest reduction in receptor-binding affinity. Most of these residues mapped to helices-1 and -4 and a loop region between helix-1 and helix-2. Two of the monoclonal antibodies that blocked TPO binding and bioactivity had determinants in helix-4. In contrast, the other three monoclonal antibodies, which were effective at blocking TPO activity but did not block initial binding of TPO to its receptor, had epitopes predominantly on helix or 3. These results suggest that TPO has two distinct receptor-binding sites that function to dimerize TPO receptors in a sequential fashion.
authors.affiliation: Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080, USA.
authors.forename: K H
authors.initials: KH
authors.lastname: Pearce
authors.forename: B J
authors.initials: BJ
authors.lastname: Potts
authors.forename: L G
authors.initials: LG
authors.lastname: Presta
authors.forename: L N
authors.initials: LN
authors.lastname: Bald
authors.forename: B M
authors.initials: BM
authors.lastname: Fendly
authors.forename: J A
authors.initials: JA
authors.lastname: Wells
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D018604:Epitope Mapping; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009154:Mutation; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins / Q000032:analysis*; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D016415:Sequence Alignment; D013926:Thrombopoietin / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; C082218:MPL protein, human; D013926:Thrombopoietin