The clathrin light chains fall into two major classes, LCA and LCB. In an intact clathrin triskelion, one light chain, of either class, is bound to the proximal segment of a heavy chain leg. Analysis of rat brain and liver complementary DNA clones for LCA and LCB shows that the two light chain classes are closely related. There appear to be several members of each class having deletions of varying length aligned at the same position. A set of ten heptad elements, characteristic of alpha-helical coiled coils, is a striking feature of the central part of each derived amino acid sequence. These observations suggest a model in which the alpha-helical segment mediates binding to clathrin heavy chains and the amino- and carboxyl-terminal segments mediate interactions with other proteins. They also suggest an explanation for the observed tissue-dependent size variation for members of each class.
The inhibitory effect of sodium arsenite and azide on asialoorosomucoid endocytosis was tested using isolated rat hepatocytes. Under either continuous flux conditions or a single synchronous wave of ligand endocytosis we confirm that azide inhibits the recycling of the receptors and we provide evidence for the involvement of thiol groups in the internalization step. In addition pretreatment of hepatocytes with azide allows us to demonstrate that receptor endocytosis proceeds independently of the presence of any specific ligand.
Recently, it has emerged that extracellular proteases have specific regulatory roles in modulating immune responses. Proteases may act as signaling molecules to activate the Raf-1/extracellular regulated kinase (ERK)-2 pathway to participate in mitogenesis, apoptosis, and cytokine production. Most reports on the role of protease-mediated cell signaling, however, focus on their stimulatory effects. In this study, we show for the first time that extracellular proteases may also block signal transduction. We show that bromelain, a mixture of cysteine proteases from pineapple stems, blocks activation of ERK-2 in Th0 cells stimulated via the TCR with anti-CD3ε mAb, or stimulated with combined PMA and calcium ionophore. The inhibitory activity of bromelain was dependent on its proteolytic activity, as ERK-2 inhibition was abrogated by E-64, a selective cysteine protease inhibitor. However, inhibitory effects were not caused by nonspecific proteolysis, as the protease trypsin had no effect on ERK activation. Bromelain also inhibited PMA-induced IL-2, IFN-γ, and IL-4 mRNA accumulation, but had no effect on TCR-induced cytokine mRNA production. This data suggests a critical requirement for ERK-2 in PMA-induced cytokine production, but not TCR-induced cytokine production. Bromelain did not act on ERK-2 directly, as it also inhibited p21ras activation, an effector molecule upstream from ERK-2 in the Raf-1/MEK/ERK-2 kinase signaling cascade. The results indicate that bromelain is a novel inhibitor of T cell signal transduction and suggests a novel role for extracellular proteases as inhibitors of intracellular signal transduction pathways.
Using high concentrations of extracellular [3H]asialoorosomucoid we obtained the steady-state level of [3H]asialoorosomucoid endocytosis by isolated hepatocytes from normal and streptozotocin diabetic rats.At the steady-state of the overall reaction, the intracellular amount of [3H]asialoorosomucoid did not change with time, the apparent overall rate of [3H]asialoorosomucoid degradation was close to that of [3H]asialoorosomucoid internalization, in both normal and diabetic rat hepatocytes.Although in diabetic cells the intracellular amount of [3H]asialoorosomucoid was about three-times lower than in normal cells, the same fraction of intracellular asialoorosomucoid was degraded per time interval by both normal and diabetic cells.The apparent first-order rate constant of steady-state degradation was about 0.01 1 min-' for both normal and diabetic cells.In diabetic rat hepatocytes, the decrease of the clearance of serum asialoglycoproteins was directly correlated to the variation of cell surface receptor number, without any modification of internalization and degradation steps.Previous studies have reported that among various metabolic disturbances, diabetes mellitus is accompanied by evident changes in the glycoconjugates of different cell membranes [l-41. So we have previously shown that the biological activity of hepatic receptor which mediates the clearance of serum asialoglycoproteins [5,6] is altered in hepatocytes from streptozotocin diabetic rats [4]. This alteration is due to a decrease of the hepatocyte capacity to bind and take up asialoglycoproteins [7,8]. The decrease of the biological activity is not correlated to that of sialic acid in membrane [9], but depends on the insulin deficiency [7].Another disturbance, due to insulin deficiency, could happen during the asialoglycoprotein degradation in the liver. Indeed, in addition to the stimulation of protein synthesis, insulin markedly decreases protein breakdown in normal hepatocytes [lo-121. In the same way, an enhanced degradation of intrahepatic and extrahepatic proteins by liver associated with diabetes has been shown [13,14], suggesting an alteration in the degradative mechanism.In the present study, we have examined the asialoglycoprotein degradation by normal and diabetic rat hepatocytes. Using high concentrations of extracellular asialoorosomucoid in order to obtain the steady-state of the overall reaction, we failed to find any discrepancy in the asialoorosomucoid degradation between diabetic and normal rat hepatocytes.
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