Mouse T lymphoma cells contain a transmembrane glycoprotein (GP85) that binds ankyrin.
In this study we have used complementary biochemical and immunological techniques to establish that the lymphoma GP85 membrane glycoprotein is a transmembrane protein with a cytoplasmic domain that binds directly to ankyrin, a molecule known to link the membrane to the cytoskeleton. The evidence supporting our conclusion that the GP85 is a transmembrane glycoprotein is as follows: (a) GP85 can be surface-labeled with Na 125I and contains wheat germ agglutinin-binding sites, indicating that it has an extracellular domain; (b) GP85 can be phosphorylated by intracellular kinases, indicating that it has an intracellular domain; and (c) GP85 can be successfully incorporated into phospholipid vesicles, indicating the existence of a hydrophobic domain in the molecule. Further studies show that GP85 displays immunological cross-reactivity with the lymphocyte Pgp-1 (differentiation-specific) membrane glycoprotein, and with the erythrocyte anion transport membrane protein, band 3. Immunocytochemical studies indicate that an ankyrin-like protein accumulates underneath the lymphoma GP85 cap structure, suggesting an association of the ankyrin-like protein and GP85. This relationship has been further confirmed by the following results of binding and reconstitution experiments: (a) purified GP85 binds directly to an ankyrin-Sepharose column; (b) purified GP85 inserts into phospholipid vesicles in both the normal (right side out) and reversed (inside out) orientation (and with only the reversed configuration permits binding of ankyrin to GP85); and (c) cleavage of GP85 with trypsin yields a 40-kD peptide fragment that is part of the cytoplasmic domain and contains the ankyrin binding site(s). Based on these findings, we suggest that the lymphoma GP85 transmembrane glycoprotein contains a cytoplasmic domain that is directly involved in linking ankyrin to the cytoskeleton. This transmembrane linkage may play a pivotal role in receptor capping and cell activation in lymphocytes.
D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000276:immunology; D017487:Ankyrins; D001798:Blood Proteins / Q000378:metabolism*; D003429:Cross Reactions; D003599:Cytoskeleton / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D008562:Membrane Glycoproteins / Q000276:immunology / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D010449:Peptide Mapping; D011485:Protein Binding; D013601:T-Lymphocytes / Q000378:metabolism / Q000648:ultrastructure*; D014407:Tumor Cells, Cultured
D001457:Anion Exchange Protein 1, Erythrocyte; D017487:Ankyrins; D001798:Blood Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins