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)(11)(12)(13)(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...

The significance of intracellular Na ÷ concentration in catecholamine secretion of cultured bovine adrenal chromaffin cells was investigated using the monovalent carboxylic ionophore monensin. This ionophore, which is known to mediate a one-for-one exchange of intracellular K ÷ for extracellular Na ÷, induces a slow, prolonged release of catecholamines which, at 6 h, amounts of 75-90% of the total catecholamines; carbachol induces a rapid pulse of catecholamine secretion of 25-35%. Although secretory granule numbers appear to be qualitatively reduced after carbachol, multiple carbachol, or Ba 2÷ stimulation, overall granule distribution remains similar to that in untreated cells. Monensin-stimulated catecholamine release requires extracellular Na ÷ but not Ca 2+ whereas carbachol-stimulated catecholamine release requires extracellular Ca 2÷ and is partially dependent on extracellular Na ÷. Despite its high selectivity for monovalent ions, monensin is considerably more effective in promoting catecholamine secretion than the divalent ionophores, A23187 and ionomycin, which mediate a more direct entry of extracellular Ca ~+ into the cell. We propose that the monensin-stimulated increase in intracellular Na + levels causes an increase in the availability of intracellular Ca 2+ which, in turn, stimulates exocytosis. This hypothesis is supported by the comparable stimulation of catecholamine release by ouabain which inhibits the outwardly directed Na ÷ pump and thus permits intracellular Na ÷ to accumulate. The relative magnitudes of the secretion elicited by monensin, carbachol, and the calcium ionophores, are most consistent with the hypothesis that, under normal physiological conditions, Na ÷ acts by decreasing the propensity of Ca2÷-sequestering sites to bind the Ca 2+ that enters the cell as a result of acetylcholine stimulation.The key role of Ca ~+ in stimulus-secretion coupling in secretory cells was first recognized by Douglas and Rubin (1). The subsequent availability of divalent cation ionophores, e.g. A23187, lasalocid (X-537A), ionomycin, prompted an extensive series of investigations of the ability of these agents to promote cellular secretion by admitting extracellular Ca 2+ into cells (2-11).A number of recent studies indicate that a rise in the cytoplasmic Na + concentration also stimulates the secretory activity of cells (12)(13)(14)(15). The Na ÷ may dislodge a small fraction of the relatively large store of intracellularly sequestered Ca ~÷ so that it becomes available to the presumptive intracellular Ca 2+ receptors, calmodulin or troponin C (16). Alternately, increased intracellular Na + may interfere with the ability of the intracellular Ca2+-sequestering mechanisms to intercept Ca 2÷ that is brought into the cytosol by secretagogue-initiated processes. Ouabain, which alters the egress of intracellular Na + by a specific inhibition of the Na+-K+-ATPase (Na ÷ pump), elevates intracellular Na ÷ levels and thereby stimulates secretion of catecholamines, acetylcholine, and insulin (17)(18)(...

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The binding of specific ligands to neutrophil cell surface receptors and the association of these receptors with the cytoskeleton may represent an essential step in activation. To identify surface proteins that are linked to the cytoskeleton during activation, neutrophil l25l-surface labeled plasma membranes were extracted with Triton X-100, and the soluble and insoluble (cytoskeleton) fractions analyzed by SDS-PAGE and autoradiography. The major cell surface proteins recruited to the cytoskeleton after activation with Con A, FMLP, zymosan-activated serum, or immune complexes possessed a relative molecular mass in the range of 80 to 13 kD. In addition to these proteins, WGA stimulates the recruitment of a 140-kD protein (GP 140) (RBC) band 4.1 and shows strong cross-reactivity with RBC anti-band 4.1 antibody. Phosphorylation of cytoskeletal proteins like 4.1 may be involved in the regulation of interactions between GP 140 and the actin-containing cytoskeleton. Unlike the C3bi receptor, GP 140 is a major surface component of unactivated PMNs, has no stoichiometrically related 95-kD subunit, and has two isoforms with pIs in the range of 6.4 to 6.6. Under conditions that result in an increased expression of the C3bi receptor (such as treatment with the Ca2" ionophore A23187), the amount of GP 140 on the PMN cell surface appears to be significantly reduced. The interaction of GP 140 with the cytoskeleton during activation suggests that GP 140 may play an important role in neutrophil functional responses. IntroductionThe association of PMN cell surface membrane proteins with the cytoskeleton is essential for processes such as shape change,