The lymphocyte‐function‐associated antigen‐1 (LFA‐1), the complement receptor type 3 (CR3) and the antigen p150,95 are cell‐surface glycoproteins. They are heterodimeric complexes, each containing a unique alpha‐subunit noncovalently associated with a common beta‐subunit. We have purified the beta‐subunit from human spleen and obtained limited peptide sequences. What appears to be the complete primary structure for the fully processed beta‐subunit was obtained by cDNA sequencing of clones from a phorbol ester (PMA) stimulated U937 cDNA library. There are five possible glycosylation sites and a transmembrane segment. The sequence contains a high level of cysteine (7.6%), with 24 of the 57 cysteine residues being found in three repeating units each with eight residues. The entire primary structure has 47% identity to a subunit of a fibronectin binding protein from chicken fibroblasts. It seems that LFA‐1, CR3 and p150,95 antigens may belong to an extended family of cell surface molecules including the fibronectin binding protein.

Our studies assessed the effects of increases in intracellular calcium concentrations [( Ca2+]i) on leukotriene synthesis and membrane translocation of 5-lipoxygenase (5LO). The calcium ionophore ionomycin and the tumor promoter thapsigargin stimulated leukotriene production and translocation of 5-lipoxygenase to the membrane. Both agents elicited prolonged rises in [Ca2+]i. Leukotriene C4 production associated with [Ca2+]i in cells stimulated with various concentrations of ionomycin and thapsigargin suggests that a threshold [Ca2+]i level of approximately 300-400 nM is required. In the absence of extracellular Ca2+, both the ionomycin- and thapsigargin-induced rises in [Ca2+]i were transient, indicating that the prolonged [Ca2+]i elevation is due to an influx of extracellular Ca2+. Addition of EGTA to the external medium before, or at different times during, the treatment with ionomycin or thapsigargin instantaneously inhibited 5LO translocation and leukotriene synthesis, indicating that Ca2+ influx plays an essential role in 5LO membrane translocation and leukotriene synthesis. No leukotriene production was detected when cells were stimulated by a physiological stimulus of leukotriene D4. The addition of 100 nM leukotriene D4 triggered peak rises in [Ca2+]i that were comparable to those achieved by the ionomycin and thapsigargin. However, the leukotriene D4 induced rise was transient and rapidly declined to a lower but still elevated steady-state level, which was attributed to Ca2+ influx. Stimulation with 100 nM leukotriene D4 for 15 s increased the cellular levels of 1,4,5-inositol triphosphate (IP3), 1,3,4-IP3, and 1,3,4,5-inositol tetraphosphate (IP4).(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment of rat basophilic leukemia cells (RBL-1) with the calcium ionophore A23187 resulted in activation of 5-lipoxygenase, as indicated by an induction of leukotriene release [Orning, L., Hammarström, S., & Samuelsson, B. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2017]. The enzyme activation was accompanied by a time-dependent association of 5-lipoxygenase to the particular fraction. When cells were lysed in the presence of 0.05-10 microM CaCl2, the soluble 5-lipoxygenase became associated with the particulate fraction. This was demonstrated by a decrease in immunoreactivities and enzymatic activities in the soluble fraction and a parallel increase in particulate-associated immunoreactivities. The particulate-bound enzyme was not active. Ca2+ induced the membrane association of 5-lipoxygenase when added into the incubation mixtures containing the membrane fraction with either the cytosolic fraction or the purified enzyme. 5-Lipoxygenase also bound to the microsomal-enriched fraction in the presence of Ca2+. Maximal membrane binding was obtained after a 1-min incubation at 4 degrees C. When a fixed amount of isolated membranes (0.2 mg of protein) and increasing cytosolic protein (0.5-4 mg) were used, a linear increase in enzyme binding was observed. The binding became saturated at 3 mg of cytosolic protein/mg of membrane protein. 5-Lipoxygenase binding to the membrane fraction was unaffected by pretreatment of the membranes with trypsin but was inhibited by treating with phospholipase A2, suggesting that phospholipids are involved.(ABSTRACT TRUNCATED AT 250 WORDS)

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Recent studies in rat basophilic leukemia cells (RBL-2H3) have shown that two pharmacological agents, ionomycin and thapsigargin, induce leukotriene C4 production and translocation of 5-lipoxygenase from cytosol to membrane, primarily by causing an influx of extracellular calcium. In the present study, we investigate the induction of these events by receptor activation. Cross-linking of high-affinity IgE receptors (Fc epsilon RI) by antigen in RBL-2H3 cells leads to leukotriene C4 production and membrane translocation of 5-lipoxygenase. As in the ionomycin-stimulated cells, leukotriene C4 production in antigen-stimulated cells is calcium-dependent since the amount of leukotriene C4 produced correlates quantitatively with the increase in intracellular free calcium concentration ([Ca2+]i). However, the increase in [Ca2+]i required for equivalent leukotriene C4 production by antigen is not as high as it is using ionomycin. In addition, no threshold [Ca2+]i level is required for leukotriene production by antigen, which is in contrast to the ionomycin stimulation that a [Ca2+]i level of 300-400 nM is required. Furthermore, antigen causes an additive increase in leukotriene C4 production in cells stimulated by the ionomycin. These results suggest that another as yet unidentified intracellular pathway acts in conjunction with Ca2+ for leukotriene synthesis in antigen-stimulated cells. Antigen stimulation causes 20-30% of the total cell 5-lipoxygenase to associate with membranes (compared with 10% in unstimulated cells) as demonstrated by enzyme activity assay and by Western Blot using antibodies to 5-lipoxygenase.(ABSTRACT TRUNCATED AT 250 WORDS)