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Analysis of protein kinase C requirement for exocytosis in permeabilized rat basophilic leukaemia RBL-2H3 cells: a GTP-binding protein(s) as a potential target for protein kinase C.

ABSTRACT: The role of protein kinase C in calcium-dependent exocytosis was investigated in permeabilized rat basophilic leukaemia cells. When protein kinase C was down-regulated by phorbol myristate acetate (1 microM for 3-6 h) or inhibited by pharmacological agents such as calphostin C (1 microM) or a protein kinase C-specific pseudo-substrate peptide inhibitor (100-200 microM), cells lost the ability to secrete in response to 10 microM free Ca2+. In contrast, a short treatment (15 min) with phorbol myristate acetate, which maximally activates protein kinase C, potentiated the effects of calcium. Biochemical analysis of protein kinase C-deprived cells indicated that loss of the Ca(2+)-induced secretory response correlated with disappearance of protein kinase C-alpha. In addition, at the concentrations effective for exocytosis, calcium caused translocation of protein kinase C-alpha to the membrane fraction and stimulated phospholipase C, suggesting that, in permeabilized cells, protein kinase C can be activated by calcium through generation of the phospholipase C metabolite diacylglycerol. The delta, epsilon and zeta Ca(2+)-independent protein kinase C isoenzymes were insensitive to phorbol myristate acetate-induced down-regulation and did not, as expected, translocate to the particulate fraction in response to calcium. Interestingly, secretory competence was restored in cells depleted of protein kinase C or in which protein kinase C itself was inhibited by non-hydrolysable GTP analogues, but not by GTP, suggesting that protein kinase C might regulate the ability of a G protein(s) directly controlling the exocytotic machinery to be activated by endogenous GTP.

SUBMITTER: Buccione R

PROVIDER: S-EPMC1137995 | biostudies-other | 1994 Feb

REPOSITORIES: biostudies-other

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Project description:Several laboratories have created rat basophil leukemia (RBL) cell lines stably transfected with the human high affinity IgE receptor (FcεRIH). More recently, humanized RBL cell lines saw the introduction of reporter genes such as luciferase (RS-ATL8) and DsRed (RBL NFAT-DsRed). These reporters are more sensitive than their parental non-reporter humanized RBL cell lines. However, no studies so far have addressed the levels of FcεRIH surface expression on humanized RBL cell lines. This is a critical parameter, as it determines the ability of these cells to be efficiently sensitized with human IgE, hence it should affect the sensitivity of the cell assay-a critical parameter for any diagnostic application. Our purpose was to assess and compare the levels of expression of the transfected FcεRIH chain in humanized RBL cell lines. We compared surface levels of FcεRIαH by flow cytometry, using a fluorescently labelled monoclonal antibody (CRA-1/AER-37) and determined receptor numbers using calibration microspheres. FcεRIαH copy numbers were assessed by qPCR, and the sequence verified. Transfection with FcεRIγH cDNA was assessed for its ability to increase FcεRIαH expression in the NFAT-DsRed reporter. While both SX-38 and RS-ATL8 expressed about 500.000 receptors/cell, RBL 703-21 and NFAT-DsRed had approximately 10- to 30-fold lower FcεRIαH expression, respectively. This was neither related to FcεRIH gene copy numbers, nor to differences in steady state mRNA levels, as determined by qPCR and RT-qPCR, respectively. Instead, FcεRIαH surface expression appeared to correlate with the co-expression of FcεRIγH. Stable transfection of NFAT-DsRed cells with pBJ1 neo-huFcεRI gamma, which constitutively expresses FcεRIγH, increased FcεRIαH chain expression levels. Levels of FcεRIαH surface expression vary greatly between humanized RBL reporter cell lines. This difference will affect the sensitivity of the reporter system when used for diagnostic purposes.

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Analysis of protein kinase C requirement for exocytosis in permeabilized rat basophilic leukaemia RBL-2H3 cells: a GTP-binding protein(s) as a potential target for protein kinase C.

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