新穎型小鼠鳥苷酸環化酶受體-G活化機制之探討

鳥苷酸環化酶（guanylyl cyclase，GC）已知可調控內生性環鳥嘌呤單磷酸核苷（cyclic guanosine monophosphate，cGMP）合成。近年來發現的小鼠鳥苷酸環化受體-G（guanylyl cyclase receptor G，mGC-G）仍然是孤兒受體，尚無已經的配體；在我們實驗室先前的研究中證實：mGC-G表現於精子且可以影響精子泳動力；我們更進一步證實，在女性生殖系統中，卵巢內的顆粒細胞和卵母細胞mGC-G也會表現。綜合以上的發現將假設建立在mGC-G藉由homo- philic-extracellular domain（ECD）的相互作用來調控分子的辨識作用和下游的訊息傳遞。因此我們建構四種穩定表現mGC-G的細胞株包含293T-pFlag-mGCG，CHO-K1-pFlag-mGCG，293T-mGCG-ECD-human IgG-Fc，以及293T-mGCG-ECD-His-myc細胞株，來證實此一假設。mGC-G這種特殊的homophilic-ECDs相互作用可以經由細胞模式和蛋白模式的酵素免疫分析法（Enzyme-linked immunoassay，ELISA）得到證實；更利用雷射共軛焦顯微鏡觀察免疫染色的結果：以anti-human IgG-Fc-FITC抗體辨認結合在293T-pFlag-mGCG細胞上的mGCG-ECD-human IgG-Fc蛋白，證實兩者的相互作用。這種mGC-G特殊的ECD-ECD domain相互作用的結合率和解離率也經由Scathard plot計算出來，Kd = 7.34×10-8 M。更重要的是mGC-G這種同質性的相互作用可以引發下游內生性cGMP的產生。簡單說來，我們的實驗證實細胞上表現的mGC-G受體可以產生相互作用，是經由這種特殊的ECD相互作用。這種homophilic-ECD相互作用可能扮演分子間辨識和訊息傳遞作用。

關鍵字

小鼠鳥苷酸環化酶受體

並列摘要

Guanylyl cyclase (GC) is known to transmit signaling by synthesizing of intracellular cyclic GMP. In mouse, guanylyl cyclase receptor G (named mGC-G) has recently been identified to be an orphan receptor with unknown ligands. Our previous report demonstrated that mGC-G prominently expresses in sperm to regulate the sperm motility. Further studies showed mGC-G was also expressed in granulosa cells and ooplasm in female ovaries. Together with our previous observations, we therefore hypothesized the homophilic-extracellular domain (ECD) interaction of mGC-G receptor in molecular recognition as well as signal transduction in cells. To test our hypothesis, we established four stable mGC-G expression cell lines, including 293T-mGC-G-Flag, CHO-K1-mGC-G-Flag, 293T-mGC-G-ECD-human IgG-Fc, and 293T-mGC-G-ECD-His-myc. The specific homophilic-ECDs interaction of mGC-G was demonstrated by protein-based Enzyme-Linked ImmunoSorbent Assay (ELISA) and cell-based ELISA model. This result is further confirmed by confocal immunostaining in 293T-pFlag-mGC-G cells by utilizing a specific anti-human IgG-Fc-FITC polyclonal antibody. The disassociation constant of the homophilic ECDdomain interaction of mGC-G was also estimated by Scatchard plot. It was estimated to be 7.34×10-8 M. Most importantly, the homophilic interaction of mGC-G receptors was capable of intracellular cGMP production. In summary, here we presented the specific homophilic-extracellular domain (ECD) interaction of mGC-G in cell-cell interaction. The homophilic-ECD interaction of mGC-G receptors on cells may play roles in molecular recognition as well as signal transduction in cells.

並列關鍵字

mouse guanylyl cyclase-G homophilic interaction cGMP

參考文獻

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新穎型小鼠鳥苷酸環化酶受體-G活化機制之探討

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