Loss of expression of human spectrin src homology domain binding protein 1 is associated with 10p loss in human prostatic adenocarcinoma.

The gene encoding human spectrin Src homology domain binding protein 1, or Hssh3bp1, which is a marker of macropinocytic vesicles and a potential regulator of macropinocytosis, co-localizes to a YAC containing chromosome 10p sequences at loci D10S89 and D10S111 that are frequently deleted in prostate tumors. Expression of Hssh3bp1 was evaluated at the protein level in 17 paired normal and malignant prostate tumor samples using the monoclonal antibody 2G8 to Hssh3bp1. These experiments demonstrated that 4/6 tumors (67%) with 10p deletion failed to express Hssh3bp1 protein compared to 5/11 (46%) tumors with intact 10p. Thus, loss of Hssh3bp1 expression is concordant with allelic loss of adjacent 10p sequences in human prostate tumors. In addition, two prostate tumor cell lines contain an exon skipping mutation in the Hssh3bp1 gene that leads to the abnormal splicing of the mRNA and loss of a portion of Abl tyrosine kinase SH3 domain binding site in the protein. These data are consistent with a role for Hssh3bp1 as a candidate tumor suppressor gene inactivated during prostate tumorigenesis.

Monoclonal antibodies to alphaI spectrin Src homology 3 domain associate with macropinocytic vesicles in nonerythroid cells.

Spectrins represent a family of membrane-associated proteins responsible for membrane flexibility and cell shape in erythrocytes, and probably in most nonerythroid cells. Spectrin functions as a tetramer consisting of two heterodimers each containing two subunits termed alpha and beta. In humans, alphaI and alphaII spectrins but not beta spectrins are characterized by the presence of an Src homology 3 (SH3) domain. As a tool to investigate the function of spectrin SH3 domains we derived several monoclonal antibodies (mAb) to the recombinant human alphaI or alphaII spectrin SH3 domain. Immunostaining using these monoclonal antibodies indicated expression of alphaI spectrin in cell bodies and alphaII spectrin in neurites of granule neurons in mouse primary cerebellar cultures. Monoclonal antibodies reactive to alphaI spectrin SH3 domain indicated expression of a protein(s) containing an alphaI-like SH3 domain in cytoplasmic vesicular-like structures in GFAP-positive cells in these cultures. In NIH 3T3 fibroblasts, these antibodies label macropinocytic vesicles. Together, these data and Western blotting results suggest expression of at least three spectrin-SH3 domain antibody-reactive proteins.

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 coated vesicles larger than 0.2 microm in diameter. Although macropinocytic vesicles are readily visualized in cultured cells by the introduction of fluorescent, water-soluble dyes into the culture medium, protein markers associated with this type of vesicles have not yet been well defined. Here, we report that human spectrin SH3 domain binding protein 1, or Hssh3bp1, associates 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 endocytosis of fluorescent dyes into the Hssh3bp1 macropinosomes and dramatically 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 Hssh3bp1 in macropinocytosis. In the macropinosomes of NIH 3T3 cells, Hssh3bp1 associates 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.