Department of Molecular Enzymology, Intercollegiate Faculty of Biotechnology of University of Gdansk and Medical University of Gdansk, Gdansk, Poland.
J Physiol Pharmacol. 2009 Oct;60 Suppl 4(Suppl 4):39-46.
Previously, using cultured human epidermal keratinocytes we have demonstrated that the activity of CRF1 receptor can be modulated by the process of alternative splicing. This phenomenon has been further investigated in the mouse corticotroph AtT-20 cell line. In the cells, transiently transfected with the plasmids coding human CRF1 isoforms, only isoforms alpha and c have shown expression on the cell membrane. Other isoforms d, e, g and h had intracellular localization with the isoform e also found in the nucleus. Co-expression of the CRF1alpha (main form of the receptor) with isoforms d, f and g prevented its expression on the cell surface resulting in accumulation of CRF1alpha inside of the cell. s expected, CRF stimulated time and dose dependent activation of CRE, CARE, AP-1 transcription elements and POMC promoter in AtT-20 cells overexpressing human CRF1alpha, while having no effect on the AP-1 transcriptional activity in cells transfected with other isoforms (d, f, g and h). However, when cells were co-transfected with CRF1alpha and CRF1e or h the CRF stimulated transcriptional activity of CRE and AP-1 was amplified in comparison to the cells expressing solely CRF1alpha; the effect was more pronounced for CRF1h than for CRF1e. In contrast, the conditioned media from the cells overexpressing CRF1e and h inhibited the CRF induced transcriptional activity in cells overexpressing CRF1alpha. Media from cells expressing CRF1h were significantly more potent that from cells transfected with CRF1e. In summary, we have demonstrated that alternatively spliced CRF1 isoforms can regulate the cellular localization of CRF1alpha, and that soluble CRF1 isoforms can have a dual effect on CRF1alpha activity depending on the intracellular vs. extracellular localization.
先前，我们使用培养的人表皮角质形成细胞证明了 CRF1 受体的活性可通过选择性剪接的过程进行调节。这一现象在鼠 corticotroph AtT-20 细胞系中得到了进一步的研究。在转染编码人 CRF1 异构体的质粒的细胞中，只有异构体 alpha 和 c 显示在细胞膜上表达。其他异构体 d、e、g 和 h 具有细胞内定位，其中异构体 e 也存在于核内。CRF1alpha（受体的主要形式）与异构体 d、f 和 g 的共表达阻止了其在细胞表面的表达，导致 CRF1alpha 在细胞内积累。预期，CRF 刺激时间和剂量依赖性地激活 CRE、CARE、AP-1 转录因子和 POMC 启动子在过度表达人 CRF1alpha 的 AtT-20 细胞中，而对其他异构体（d、f、g 和 h）转染的细胞中的 AP-1 转录活性没有影响。然而，当细胞共转染 CRF1alpha 和 CRF1e 或 h 时，CRF 刺激的 CRE 和 AP-1 的转录活性与仅表达 CRF1alpha 的细胞相比被放大；对于 CRF1h，这种效应比 CRF1e 更为明显。相比之下，过度表达 CRF1e 和 h 的细胞的条件培养基抑制了过度表达 CRF1alpha 的细胞中 CRF 诱导的转录活性。表达 CRF1h 的细胞的条件培养基比转染 CRF1e 的细胞的条件培养基更有效。总之，我们已经证明了选择性剪接的 CRF1 异构体可以调节 CRF1alpha 的细胞定位，并且可溶性 CRF1 异构体可以根据细胞内与细胞外定位对 CRF1alpha 的活性产生双重影响。
