Interaction of insulin-like growth factor binding protein-4, Miz-1, leptin, lipocalin-type prostaglandin D synthase, and granulin precursor with the N-terminal half of type III hexokinase

Insulin-like growth factor binding protein-4, Miz-1, leptin, prostaglandin D synthase, and granulin precursor were identified as proteins interacting with the N-terminal half of mammalian Type In. hexokinase (HKIII) in the ye ast two-hybrid method. These interactions were confirmed by in vitro bindin g studies. All five of these proteins, and their mRNAs, were present in PC1 2 cells, as shown by immunoblotting and RT-PCR, respectively. All were coim munoprecipitated from PC12 extracts with an antibody against HKIII, brit no t with anti-Type I hexokinase. Moreover, all of these proteins were coimmun oprecipitated using antileptin as precipitating antibody, indicating the ex istence of a macromolecular complex including these five proteins and HKIII . Transfection of M+R 42 cells with HKIII-green fluorescent protein (GFP) r eporter constructs gave a diffuse intracellular fluorescence. Cotransfectio n with leptin or Miz-1 resulted in distinctly different localization of the HKIII-GFP fusion protein, at intracellular sites coincident with localizat ion of leptin-GFP or Miz-1-GFP reporter constructs. (C) 2000 Academic Press .