發現G蛋白beta次元體3之選擇性剪切變異及其在信息傳導上的角色

Abstract

生物體中有許多生化機制被證實是經由G蛋白的訊息傳導進而調控。目前有許多大型的流行病學領域之研究已提供初步的證據，指出G蛋白複合體中的β次元體第三亞型（G protein beta subunit 3, GNB3）可能與高血壓、心血管疾病、中風、糖尿病、肥胖、腎臟病、憂鬱症、偏頭痛、暈眩症等疾病相關。在我們實驗室先前的研究也指出，GNB3在肥胖患者身上的表現量明顯高於一般人，但是它與這些疾病的確切相關途徑仍未確定。基本上，G蛋白的下游訊息傳導主要是由α次元體以及βγ所組成的異次元體分別活化下游的信息傳遞因子。在生物化學領域中，也有少數的研究發表關於GNB3的生化活性及其生理功能，主要是由Rosskopf Dieter所屬之德國團隊，分別在1998及2003年所發現之選擇性剪切變異（Alternative splicing variant）GNB3s及GNB3s2的研究中，在細胞試驗中證實此二種選擇性剪切變異不僅可能造成蛋白質結構的改變，更影響其本身與G蛋白α及γ次元體之交互作用和結合能力，進一步還會改變下游PLCβ2（Phospholipase C beta 2）和MAPK（Mitogen-activated protein kinase）的活性，可見GNB3的確在信息傳遞上扮演著重要的角色。 為了釐清GNB3是否與肥胖有相關性，我們從敏盛醫院和恩主公醫院招募數個接受腸縮減手術的肥胖患者，並詢問其家屬是否願意一起加入本實驗研究。我們採集同意者的血樣進行進一步的純化和分析，將受試者的GNB3基因進行PCR放大並且定序分析其基因變異。我們發現這些肥胖家族成員都含有不只一種GNB3的選擇性剪切變異，且有兩個出現頻率非常高的單一核酸多形性G814A和C825T。對於這個現象，我們進一步分析不同的基因變異是否造成GNB3的生理活性改變。 利用細胞表現的系統，我們用腺苷酸環化酶激活劑(forskolin)直接活化腺苷酸環化酶(Adenylyl cyclase)，再將GNB3基因轉染(transfect)進細胞中表現。細胞中的環腺苷酸(cAMP)量會因為加入10μM forskolin活化而顯著升高，但是否轉染GNB3基因對細胞的環腺苷酸沒有顯著的影響。因此我們轉而測試另外一個信息傳遞的成員，有絲分裂活化蛋白激酶(MAPK)。細胞受到不同時間的forskolin刺激，會影響有絲分裂活化蛋白激酶的磷酸化(pMAPK)程度。沒有轉染GNB3的細胞受到forskolin的刺激會很快的增加pMAPK的量，但有轉染GNB3的細胞則會比較平緩的活化pMAPK。如果我們在細胞轉染先前純化出的不同GNB3基因變異型，可以發現不同的變異型會受到forskolin不同程度的活化。刺激前後的pMAPK量的改變從1.10到17.51倍不等。因此我們推測，肥胖家族的確有許多選擇性剪切變異存在，這些變異可能會經由改變細胞中MAPK的活化，進一步影響生理機制而造成肥胖。

Heterotrimeric G-proteins have been proved to be involved in various signaling pathways via heptahelical receptors. Since these groups of G protein trimeric complex may play some important roles in signal transduction, we speculate that they may affect the metabolism in vivo. The previous studies in our laboratory found that weight change of patients receiving bariatric surgery is strongly associated with the guanine nucleotide-binding protein, beta polypeptide 3 (abbreviated as GNB3) gene expression level. It is proved that G proteins usually form beta-gamma heterodimer and serve as effectors to regulate downstream signaling. Previous researches have also pointed out that the GNB3 gene is prone to have single nucleotide polymorphisms (SNPs) and result in several different alternative splicing forms. Lots of epidemiological researches had showed that GNB3 C825T is strongly associated with hypertension, obesity, diabetes, and many other metabolic disorders. Also, there was some biological data show this polymorphism is associated with alternative splicing of exon 9. It may generate GNB3-s and GNB3-s2 two splicing variants and enhance G protein signaling. But the actual mechanism that GNB3 involved in is still unclear. In order to figure out what roles GNB3 played and what components may be affected by it, we established series of experimentations. First, we recruited the patients with obesity (BMI>28) and receiving bariatric surgery in En-Chu-Kong hospital and Min-Sheng Hospital. We also invite their family members who are willing to join our research. Then we collected their blood samples and used Epstein-Barr virus to infect into B lymphoblastoid cell to establish human biobank refers to each patient’s genomic background. Second, we extracted these patients’ RNA from EBV infected cell lines or direct from their blood sample. And doing reverse transcription to get cDNA to further PCR amplification. The PCR primer pair is designed with two restriction enzyme sites, EcoRI and XhoI, to further cloning procedure. After ligasing with pcDNA 3.0 MycA vector, these clones was send to DNA sequencing center to confirm their SNPs or splicing forms. The results showed that G814A and C825T are two common SNPs in these subjects, and all of these genes are prone to generate alternative splicing products. We got ten different splicing variants and wild type to do subsequent bioassay. Since these splicing forms discovered from obese objects seem to be very different from wild type, we are interested in whether the biological functions of these variants will change or not. So we firstly examined the downstream signal, cyclic AMP, by using luciferase construct response for cAMP level. It seems that GH3, HepG2 cells treat with forskolin will enhance cAMP level, but the GNB3 gene had no effect on cAMP level. Therefore, we test another downstream component that had been published to associate with G protein signaling, that is, MAP kinase. The MAP kinase activity was detected by anti-phosphorylated p38 MAP kinase antibody of p38 MAPK kit. The MAPK phosphorylation level is activated when cells treat with forskolin. Over express GNB3 gene will delay the activation. Different variants have distinguished response of forskolin. The effects of MAPK phosphorylation are activated in different fold changes from 1.10 to 17.51 times of activation. Thus, we can surmise that GNB3 gene variants are exited in obese families and play an important role on MAPK signaling.