FFAR2 N-糖基化修饰对受体功能的影响及与心肌肥大的关系

In our previous study about the relationship between free fatty acid receptor 2(FFAR2) and cardiac hypertrophy, we found out that FFAR2 was N-glycosylated in primary rat cardiomyocytes and the N-glycosylation level was significantly upregulated during phenylephrine-induced cardiomyocyte hypertrophy. But the significances of this modification still needs further investigation. It is known that N-glycosylation may affect the function of some G-protein-coupled receptors including membrane localization of receptors, affinity of the binding of ligands and receptors, stabilization of receptors and signal transduction. Based on our previous study that the expression of FFAR2 was increased in in vivo pressure overload-induced cardiac hypertrophy and in vitro phenylephrine-induced cardiomyocyte hypertrophy, and FFAR2 agonist or overexpression could induce cardiomyocyte hypertrophy, we hypothesized that the N-glycosylation of FFAR2 might promote its membrane localization and increase the stabilization of the receptor, and enhance the signal transduction of the receptor, which play an important role in the development of cardiac hypertrophy. In this project, we will adopt site mutation and other methods to clarify the effects of FFAR2 N-glycosylation on receptor function and the relationship between this modification and cardiac hypertrophy in in vivo and in vitro hypertrophic models. It is possible to provide some useful information for drug development targeting FFAR2.

我们在研究游离脂肪酸受体2(FFAR2)与心肌肥大关系时发现，FFAR2存在N-糖基化修饰，并且肥大诱导剂苯肾上腺素可显著提高N-糖基化修饰水平，然而这一修饰的意义并不清楚。已知N-糖基化修饰可影响某些G-蛋白偶联受体的功能，包括受体的膜定位、配体与受体的亲和力、受体的稳定以及信号转导等。结合前一课题的研究结果，即心肌肥大时FFAR2表达增加，FFAR2激动剂或者其过表达均可诱导心肌细胞肥大，我们提出假说：FFAR2的N-糖基化修饰可能促进其膜定位，增加受体的稳定，增强受体信号转导，从而在心肌肥大过程中扮演重要角色。本课题拟在体内外心肌肥大模型中，采用位点突变等方法阐明FFAR2的N-糖基化修饰对其功能的影响，以及这一修饰与心肌肥大的关系。本课题的研究可提供心肌肥大发生的新机制，并且有可能为基于FFAR2为靶点的药物开发提供参考。

游离脂肪酸受体-2（FFAR2，也叫GPR43）是丙酸等短链脂肪酸激活的G蛋白偶联受体。课题组在研究FFAR2促进心肌肥大时发现，FFAR2条带位置远远高于预测分子量，采用内切糖苷酶PNGase F处理蛋白样品可降低条带位置，提示存在N-连接糖基化修饰。软件预测的N糖基化位点151和167进行突变，分别获得野生型FFAR2（WT）、FFAR2（N151Q）、FFAR2（N167Q）和双突变的FFAR2（N151Q, N167Q）。两个位点的突变均可使FFAR2条带位置下移，但以FFAR2（N151Q）的影响更大。以细胞內钙释放及ERK1/2磷酸化作为评价FFAR2受体功能，与野生型相比，151位点突变显著抑制激动剂诱导的细胞内钙释放和ERK1/2的磷酸化，而167位点突变影响较小，双突变几乎完全抑制以上反应。进一步地，在激动剂存在的情况下，FFAR2（WT）促进肥大基因ANF和BNP表达上调，心肌细胞表面积也明显增大，而FFAR2（N167Q）诱导肥大基因表达上调和细胞表面积增大的作用减弱，FFAR2（N151Q）减弱的作用更大；与无激动剂处理相比，双突变FFAR2（N151Q, N167Q）几乎不能引起肥大指标的上升。接着，我们研究了糖基化修饰对促进肥大的转录因子GATA4和STAT3的功能影响。FFAR2（WT）可明显提高GATA4和STAT3磷酸化水平，报告基因分析两者转录活性也显著升高，而突变质粒作用较弱，尤以151位点的突变削弱作用明显。以上结果表明151和167位点均可发生N糖基化修饰，且以151位点的修饰对受体功能的影响更为显著，也是其诱导心肌肥大的重要条件。我们的研究提示，抑制FFAR2的N糖基化可能是心肌肥大防治的新策略。