Human spectrin Src homology 3 domain binding protein 1 regulates macropinocytosis in NIH 3T3 cells

Macropinocytosis is an endocytic process that occurs through non-clathrin c oated vesicles larger than 0.2 mum in diameter. Although macropinocytic ves icles are readily visualized in cultured cells by the introduction of fluor escent, water-soluble dyes into the culture medium, protein markers associa ted with this type of vesicles have not yet been well defined. Here, we rep ort that human spectrin SH3 domain binding protein 1, or Hssh3bp1, associat es with macropinosomes in NIH 3T3 fibroblasts, Hssh3bp1 macropinosomes are heterogeneous in morphology and size, do not endocytose transferrin and are resistant to brefeldin A treatment. Cytochalasin D, and wortmannin block e ndocytosis of fluorescent dyes into the Hssh3bp1 macropinosomes and dramati cally affect their morphology. Overexpression of Hssh3bp1-green fluorescent protein abolished fusion of vesicles resulting in a decreased endocytosis of fluorescence dyes, thus suggesting a potential regulatory role of Hssh3b p1 in macropinocytosis. In the macropinosomes of NIH 3T3 cells, Hssh3bp1 as sociates with a 200-kDa protein that crossreacts with a monoclonal antibody to the erythroid alpha -spectrin SH3 domain. Thus macropinosomes in cells may contain a spectrin-like protein.