A T-Lymphoma Transmembrane Glycoprotein Linked to the Cytoskeletal Protein, Fodrin (gp180) is

A major mouse T-lymphoma surface glycoprotein (gp180) has been identified by labeling cells with 1251 and [3H]glucosamine. After ligand-induced receptor patching and/or capping, the amount of gp180 in the membrane-associated cytoskeleton fraction increases in direct proportion to the percentage of patched/capped cells. There is a parallel increase in the amount of fodrin in the membrane-associated cytoskeleton fraction. Evidence is presented that gp180 is the same as or very similar to the T-lymphocyte-specific glycoprotein T-200. An immunobinding assay of Nonidet P-40-solubilized plasma membrane selectively co-isolates gp180 and fodrin. After induction of receptor rearrangement, double-label immunofluorescence reveals that fodrin accumulated directly beneath gp180 patches and caps. Membrane extraction with Triton X-114 followed by sucrose gradient centrifugation permits isolation of a gp180-fodrin complex with a 1:1 molar ratio and sedimentation coefficient(s) of approximately 20. This complex remains stable during isoelectric focusing and exhibits a pl in the range of 5.2-5.7. On the basis of our results we conclude that gp180, an intergral membrane glycoprotein, and fodrin, a component of the membrane-associated cytoskeleton, are closely associated into a complex. Furthermore, we contend that, through fodrin's association with actin, this complex is of functional significance in ligand-induced patching and capping of gp180. We also propose that, through lateral interactions in the plane of the membrane, the gp180-fodrin complex might be responsible for linking other surface receptors to the intracellular microfilament network during lymphocyte patching and capping. The involvement of contractile microfilaments in the redistribution of cell surface receptors during patching and capping was first described by Taylor et al. in 1971 (1). Since then, numerous reports have confirmed this observation for a variety of different surface receptors of mouse and human lymphocytes (2, 3). Double-label immunofluorescence microscopy reveals that cytoplasmic actin and myosin accumulate directly beneath patches and caps; that is, beneath aggregated surface receptors (4-7). More conclusively, isolation of the plasma membrane (PM) t and associated cytoskeletal elements with non-ionic detergents demonstrates that, during capping, the surface receptors form a specific association with the actin-containing cytoskeleton (8, 9). However, the nature of the linkage between membrane surface receptors and the Abbreviations used in this paper." IEF, isoelectric focusing; NP-40, Nonidet P-40; PBES, phosphate-buffered Earle's balanced salt solution; PM, plasma membrane. cytoskeleton is not understood. Recently, analogs to proteins of the erythrocyte membranecytoskeleton complex (such as spectrin, ankyrin, and glycophorin) have been identified in nonerythroid cells (10-14). Two distinct, spectrin-like proteins have been identified: fodrin (15-18) isolated from brain; and TW 260/240 (16, 17) isolated from epithelial cells of the intestinal brush border. Like spectrin ( 19-21), each of these proteins has one 240,000mol-wt subunit and a second subunit of differing molecular weight: 220,000 for spectrin (22); 235,000 for fodrin (15-18); and 260,000 for TW 260/240 (16, 17). Immunological assays and peptide mapping data indicate that the 240,000-mol-wt subunit of spectrin, fodrin, and TW 260/240 are very similar (15-17, 23, 24). In addition, all three proteins bind calmodulin and actin (15-17). In the past several years, we have studied receptor patching and capping in the mouse T-lymphoma cell line, BW 5147 THE JOURNAL OF CELL BIOLOGY VOLUME 101 AUGUST 1985 477-487 477 © The Rockefeller University Press 0021-9525/85/08/0477/I I $I .00 on O cber 0, 2017 jcb.rress.org D ow nladed fom (25-32). The major advantages of these cells are the ease of PM isolation (33) and the existence of detailed biochemical and serological characterization of several of the cell surface glycoproteins: T-200, a major T-lymphocyte-specific glycoprotein; Thy1, a common T-lymphocyte surface marker; and viral glycoprotein gp 69-71, a known surface component of lymphoma cell membranes (29, 31, 34). Previous studies showed that a number of different membrane proteins are associated with the cytoskeleton in murine cells (35, 36). In this study, we chose to investigate the relationship between surface membrane receptors and underlying cytoskeletal elements during receptor patching and capping. Biochemical and immunological analyses of isolated PM and the membrane-associated cytoskeleton indicate a close interaction between fodrin, a component of the membrane-associated cytoskeleton, and gpl80, a cell surface glycoprotein. Furthermore, our double-immunofluorescence data also show that intracellular fodrin appears to be accumulated preferentially under gp 180 patched/capped structures. Analysis of the unchallenged lymphoma PM, using a selective Triton X-114 extraction method followed by sucrose gradient centrifugaLion, reveals that gpl80 and fodrin are tightly associated into a complex with a stoichiometry of 1:1 and a sedimentation coefficient of ~20. Further analysis of the complex by isoelectric focusing (IEF) indicates that gp 180 and fodrin remain in a stable complex that exhibits a pI in the range of 5.2-5.7. We therefore propose that this structural interaction between gpl80 and fodrin is important in the functional association of the PM and the cytoskeleton during receptor patching and capping. MATERIALS AND METHODS