臺灣博碩士論文加值系統

細胞內的cGMP扮演次級訊號 (second messenger) 的角色，參與許多重要的生理功能，包括：血管平滑肌的鬆弛、抑制血小板凝集及電解質的平衡等等。在哺乳類細胞中，cGMP由兩大類的guanylyl cyclases (GCs) 所產生：一類是存在細胞質的水溶型GC，另一類是存在細胞膜上的膜型GC。膜型GC到目前為止在哺乳類細胞被發現有7個成員，分別依發現順序命名從GC-A到GC-G。最近我們實驗室從小鼠睪丸發現第七個成員GC-G，然而GC-G不同轉錄產物之組織表現及其活性的調節機制還不甚了解。為了加速對GC-G功能性研究，本實驗室想要建立一個膜型GC-G的顯性抑制 (dominant-negative) 蛋白。首先將催化部位中高度保留的天門冬胺酸 (Asp-950) 突變成丙胺酸 (alanine) 後，於細胞表達此顯性抑制蛋白後發現能夠降低50 % 由細胞內野生型 (wild-type) GC-G所產生的cGMP濃度。因此，對於未來GC-G的研究上，此顯性抑制蛋白將能夠提供一個很好的功能性分析工具。另外，我還發現GC-G有一個比較短的轉錄產物能夠轉譯成另一個新型的水溶型GC-G，命名為kinase-like domain containing soluble GC-G (ksGC-G)。利用反轉錄聚合酶鏈反應 (reverse transcriptase-PCR) 分析，發現ksGC-G廣泛地表達於不同的組織當中。更進一步，我發現ksGC-G能夠和已知由nitric oxide (NO) 調節的水溶型GC (sGC) 有交互作用，這也暗示了ksGC-G可能扮演許多與sGC類似的生物學角色。綜觀我們的研究，這是第一次發現GC-G可能藉由兩種以上不同的蛋白型式來執行其生物功能。

Intracellular second-messenger cGMP is involved in the regulation of a broad spectrum of physiological functions, including relaxation of vascular smooth muscle, repression of platelets coagulation, and electrolytes homeostasis. In the mammal, cGMP is synthesized by two major classes of enzymes: the soluble and the membrane-associated receptor guanylyl cyclases (GCs). To date, seven isoforms of receptor GCs have been described in mammals, termed GC-A to GC-G in the order of their discovery. We recently identified receptor GC-G from mouse testis. However, the expression of different GC-G transcripts and the regulation of their encoded products are largely unknown. To facilitate the functional studies, we first generate a dominant-negative (DN) mutant of receptor GC-G by introduction of an alanine (D950A) at a well-conserved Asp-950 critical for the GC activity. When overexpressed, the GC-G-D950A mutant resulted in an inactive cyclase that could form a complex and suppress a greater than 50% production of overall cGMP by the GC-G-WT protein. Therefore, this DN mutant will serve as a valuable tool to further dissect the function of cGMP signaling mediated by receptor GC-G. In addition, a shorter transcript encoding a new type of soluble GC-G, designated as kinase-like domain containing soluble GC-G (ksGC-G), was found in a wide range of tissues. Most interestingly, the ksGC-G activity could interact with nitric oxide (NO)-regulated sGC subunits, which has been implied in a variety of biological processes. Together, our results demonstrated, for the first time, that GC-G is a multi-functional protein through action by at least two distinct transcripts.

Abstract 2
摘要 3
Abbreviations 4
Table of Contents 5
Introduction 7
Research Purpose and Strategies 10
Materials and Methods 11
- Construction of plasmids 11
- Tissue RNA extraction and cDNA retrotranscription 11
- Cell culture and transfection 11
- Establishment of stable cell line 12
- Flow cytometry 12
- Immunoprecipitation 13
- Western-blot analysis 13
- Determination of protein concentration 13
- cGMP activity assay 14
Results and Discussion 15
-Receptor form of GC-G 15
-Soluble form of GC-G 18
References 23
Tables and Figures 27
- Fig.1. Intracellular regulation of membrane GC. 27
- Fig.2. A dominant negative (DN) mutant of GC-G. 28
- Fig.3. GC-G DN mutant is an inactive cyclase. 29
- Fig.4. GC-G DN mutant blocks GC-G WT activity. 30
-Fig. 5. CHO-K1 CMV-3 GC-G DN stable cell line. 31
- Fig. 6. Receptor GC-G is not regulated by PAK. 32
- Fig. 7. Primers specific for receptor GC-G or soluble GC-G (ksGC-G). 33
- Fig. 8. Tissue expression profile of membrane and soluble GC-G by RT PCR. 34
- Fig. 9. The ksGC-G cDNA clones. 35
- Fig. 10. Prediction of endogenous ksGC-G expression. 36
- Fig. 11. RT-PCR for putative ksGC-G (A2) transcript. 37
- Fig. 12. ksGC-G is capable of forming homodimers. 38
- Fig. 13. Deletion constructs of ksGC-G (A1), (B), and (C). 39
- Fig. 14. ksGC-G (A1), (B), and (C) forms contain no cyclase activity. 40
- Fig. 15. ksGC-G does not play a dominant negative role on receptor GC-G. 41
- Fig. 16. ksGC-G can not interact with PAK and regulated by it. 42
- Fig. 17. ksGC-G could interact with sGC subunits. 43
- Table 1. Primers used for expression plasmid constructs 44
- Table 2. Primers used for RT-PCR
- Table 3. Expression plasmids generated in this thesis 45
46

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