EXPRESSION OF IPLA2B AS FUSION PROTEIN W/ EGFP PROVIDES EVIDENCE FOR ITS

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定为研究者机构。胰岛b细胞和胰岛素瘤细胞表达参与信号转导的VIA组Ca 2+非依赖性磷脂酶A2（iPLA 2b）。iPLA 2b的抑制导致抑制，过表达导致促分泌素刺激的胰岛素分泌的放大。免疫荧光分析揭示了在用促分泌素刺激的INS-1细胞的核周区域中iPLA 2b的积累。在此，为了确定iPLA 2b的特异性亚细胞靶标，将其在INS-1细胞中表达为与EGFP的融合蛋白（fp），其连接在iPLA 2b的N-末端（fpC 2）或C-末端（fpN 2）。iPLA 2bas a fp的表达允许通过监测EGFP荧光在细胞中跟踪iPLA 2b，并避免抗体的潜在非特异性亲和力。fpC 2和fpN 2稳定转染的细胞比单独转染EGFP cDNA的对照细胞表达更高的iPLA 2b活性。这表明将EGFP添加到iPLA 2b不会干扰其催化活性。EGFP和细胞器追踪器的双重荧光监测揭示了iPLA 2b在受刺激的fpN 2的高尔基体和ER中积累，但不在表达fpC 2的细胞中积累。这些观察结果表明，iPLA 2b经历翻译后修饰，并且这通过使用针对EGFP或iPLA 2b的抗体的免疫亲和性分析来证实。此外，纯化的iPLA 2b的质谱分析揭示了其中可变数目的N-末端残基已被去除的iPLA 2b变体。这些发现表明，iPLA 2b经历内源性蛋白水解过程，刺激b细胞导致iPLA 2b变体重新分布到不同的亚细胞区室，

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Pancreatic islet b-cells and insulinoma cells express a Group VIA Ca2+ -independent phospholipase A2 (iPLA2b) that participates in signal transduction. Inhibition of iPLA2b leads to suppression and overexpression leads to amplification of secretagogue-stimulated insulin secretion. Immunofluorescence analyses have revealed an accumulation of iPLA2b in the perinuclear region of INS-1 cells stimulated with secretagogues. Here, to determine specific subcellular target(s) of iPLA2b, it was expressed in INS-1 cells as a fusion protein (fp) with EGFP, attached at either the N-terminus (fpC2) or C-terminus (fpN2) of iPLA2b. Expression of iPLA2bas a fp allowed iPLA2b tracking in the cell by monitoring EGFP fluorescence and avoids potential non-specific affinities of antibodies. Both fpC2 and fpN2 stably-transfected cells express higher iPLA2b activity than control cells transfected with EGFP cDNA alone. This indicates that adding EGFP to iPLA2b does not interfere with its catalytic activity. Dual fluorescence monitoring of EGFP and organelle Trackers reveals that iPLA2b accumulates in the Golgi and ER of stimulated fpN2, but not, fpC2 expressing cells. These observations suggest that iPLA2b undergoes post-translational modifications, and this is confirmed by immunoaffinity analyses using antibodies against EGFP or iPLA2b. Further, mass spectrometric analyses of purified iPLA2b reveals iPLA2b variants from which variable numbers of N-terminal residues have been removed. These findings suggest that iPLA2b undergoes endogenous proteolytic processing and that stimulation of b-cells causes redistribution of iPLA2b variants to different subcellular compartme