TI  - Mutational analysis of <prot>thrombopoietin</prot> for identification of receptor and
      neutralizing antibody sites.
PG  - 20595-602
AB  - <prot>Thrombopoietin</prot> (<prot>TPO</prot>) is a hematopoietin important for megakaryocyte
      proliferation and production of blood platelets. We sought to characterize
      how  <prot>TPO</prot>  binds and activates its receptor,  <prot>myeloproliferative leukemia
      virus receptor</prot>. The <prot>erythropoietin</prot>-like domain of <prot>TPO</prot> (TPO1-153) has been
      fused to the <prot>gIII</prot> 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 <prot>TPO</prot> receptor and five anti-<prot>TPO1</prot>-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 <prot>TPO</prot> binding and bioactivity had determinants in
      helix-4. In contrast, the other three monoclonal antibodies, which were
      effective at blocking <prot>TPO</prot> activity but did not block initial binding of
      <prot>TPO</prot> to its receptor, had epitopes predominantly on helix or 3. These
      results suggest that <prot>TPO</prot> has two distinct receptor-binding sites that
      function to dimerize <prot>TPO</prot> receptors in a sequential fashion.
AD  - Department of Protein Engineering, Genentech, Inc., South San Francisco,
