Acylation of the lymphoma transmembrane glycoprotein, GP85, may be required for GP85-ankyrin interaction.

The lymphoma plasma membrane glycoprotein, GP85, is a transmembrane glycoprotein that binds directly to ankyrin, a molecule known to link the plasma membrane with the underlying cytoskeleton. In this study, we have demonstrated that palmitic acid is incorporated into GP85 in vivo and that the amount of palmitic acid incorporated is greatly stimulated during lymphoma cap formation. The majority of the incorporated palmitic acid appears to be strongly linked to GP85 since it is not dissociated by strong detergents (e.g. sodium dodecyl sulfate) or by chloroform/methanol extraction, but is labile to alkaline or acid hydrolysis. Furthermore, we have established that deacylation of GP85 (i.e. removal of the palmitic acid moiety from GP85 by 1 M hydroxylamine treatment) significantly reduces the binding affinity between GP85 and ankyrin, and reacylation of GP85 restores the binding affinity. These findings suggest that fatty acid acylation of GP85 by palmitic acid may be required for the stable attachment of the cytoskeleton to the lymphoma plasma membrane.

Lymphoma protein kinase C is associated with the transmembrane glycoprotein, GP85, and may function in GP85-ankyrin binding.

In this study, several complementary techniques have been used to investigate the involvement of a protein kinase C (PKC) molecule in the plasma membrane-cytoskeleton interactions that occur in mouse T-lymphoma cells. Our data indicate that the lymphoma plasma membrane contains a 78-kDa polypeptide that exists in a complex with one of the major transmembrane glycoproteins, GP85 (a wheat germ agglutinin-binding protein). This membrane-associated 78-kDa protein appears to have PKC-like properties based on the following criteria: 1) it cross-reacts with a specific antibody raised against brain PKC; 2) it has a pI of 5.6-5.8, which is similar to that of the PKC described previously in other cell types; and 3) it displays characteristic PKC enzymatic activity by phosphorylating histone H1 in a Ca2+- and phospholipid-dependent manner. Double immunocytochemical staining experiments reveal that the lymphoma PKC-like molecules translocate from the cytoplasm to the cell membrane and accumulate directly underneath receptor capped structures following addition of various ligands. Studies we have done to identify the cellular substrate(s) of the lymphoma plasma membrane-associated PKC have shown that GP85 is preferentially phosphorylated in isolated membrane preparations following addition of the PKC activator, TPA (phorbol-12-O-tetradecanoyl-phorbol 13-acetate), but not the biologically inactive TPA analogue, 4 alpha-PDD (4 alpha-phorbol 12,13-didecanoate). In addition, we have found that GP85 can be phosphorylated by purified brain protein kinase C. Analysis of the resulting phosphoamino acids indicates that phosphorylation of GP85 occurs primarily at serine residues, occurs in minor amounts (approximately 5%) at threonine residues, and does not occur at tyrosine residues. These data indicate that the lymphoma GP85 is a substrate for PKC. Furthermore, we have established that phosphorylation of GP85 by PKC enhances its binding affinity with the membrane linker molecule, ankyrin. These findings suggest that PKC-mediated phosphorylation of GP85 may be an important part of the lymphoma plasma membrane-cytoskeleton interaction.

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.

Lymphoma Thy-1 glycoprotein is linked to the cytoskeleton via a 4.1-like protein.

In this study we have found that the phosphoprotein doublet of 68,000 and 65,000 daltons (68/65 kD) in mouse T-lymphoma cells shares several structural and functional similarities with erythrocyte band 4.1. Our evidence for identifying the 68/65-kD doublet as a lymphoma 4.1-like protein is as follows: it displays an immunological cross-reactivity with anti-erythrocyte band 4.1 antibody; it exhibits a Svedberg unit of sedimentation coefficient of 4 S; it is phosphorylated in the presence of phorbol ester (phorbol-12-O-tetradecanoylphorbol-13-acetate) and its phosphorylation requires Ca2+; it is phosphorylated primarily at serine residues; and it can bind directly to fodrin (a spectrin-like actin-binding protein). In addition, this lymphoma 4.1-like protein can be both colocalized and coisolated with the major T-lymphocyte-specific glycoprotein, Thy-1 (gp 25). Therefore, all of these results strongly suggest that the lymphoma 4.1-like protein (68/65-kD doublet) may play a pivotal role in linking the Thy-1 (gp 25) glycoprotein to fodrin which, in turn, binds to the actin filaments that are responsible for recruiting Thy-1 antigens into cap structures.

Thiol-specific probes indicate that the beta-chain of platelet glycoprotein Ib is a transmembrane protein with a reactive endofacial sulfhydryl group.

We used two membrane-permeable fluorescent reagents, monobromobimane and N-[[5-(dimethylamino)-1-naphthalenyl]sulfonyl]aziridine (N-dansylaziridine), and one membrane-impermeable fluorescent probe, monobromo(trimethylammonio)bimane, all three of which react selectively with protein thiols, to assess the presence of reactive sulfhydryls in the platelet glycoprotein Ib (GPIb) molecule and establish the topology of any GPIb-reactive thiols in the platelet membrane. Intact platelets were reacted with 1-10 mM monobromobimane or monobromo(trimethylammonio)bimane or 50-100 microM N-dansylaziridine for 30-60 min at 37 degree C. The platelets were then washed, solubilized in 1% Triton X-100, and analyzed by nonreduced-reduced polyacrylamide gel electrophoresis either directly or indirectly after immunopurification of GPIb. Monobromobimane and N-dansylaziridine labeled GPIb beta but not GPIb alpha in intact platelets. This labeling could be inhibited by pretreating the platelets with either N-ethylmalemide or p-(chloromercuri)benzenesulfonic acid, confirming the specificity of these probes for thiol groups. Monobromo(trimethylammonio)bimane, the membrane-impermeable reagent, did not label GPIb beta in intact platelets. However, it did label GPIb beta in sonicated platelets, indicating that the thiol group of GPIb beta occupies an intracellular location. Since the carbohydrate moiety of GPIb beta can be labeled from the outside of intact platelets with membrane-impermeable reagents, we conclude that GPIb beta has a transmembrane orientation.