INVESTIGATION OF SIGNAL TRANSDUCTION AND ANTI-PROLIPERATIVE ACTIONS OF ANGIOTENSIN II TYPE 2 PECEPTOR

具有七个跨膜区结构的受体通过激活特定的G-α蛋白来启动信号转导。本项目的目的是通过检测1型血管紧张素受体(AT1)和2型血管紧张素受体(AT2)的G-α和蛋白质识别谱来了解这两种受体在信号转导中的最初步骤。将嵌合的GαS蛋白的受体结合区改变为含有Gα蛋白4个主要家族(GαQ、GαI、Gα12、GαS)的氨基酸，将其与AT1或AT2受体共转染COS细胞，并用血管紧张素II(Ang II)刺激。用酶免疫分析法检测细胞裂解产物中cAMP的变化。在GαQ家族中，AT1与Gα11/GαS、Gα14/GαS、Gα16/GαS共转染后，激动剂刺激后cAMP显著升高，而与GαS共转染后未见明显变化。为了证实这些发现，血管紧张素Ⅱ在[35S]GTPγS存在下刺激血管平滑肌细胞膜部分，然后用抗GαQ/11、Gα14和GαS的抗体免疫沉淀G蛋白。对Gα12蛋白和GαI家族蛋白的嵌合G蛋白的进一步研究表明，AT1受体识别Gα12、Gαi1/i2、Gαz、Gαo，而两种受体都识别Gαi3。这些结果提示AT1和AT2受体的Gα蛋白识别图谱，这对于理解激素Ang II介导的细胞反应的全谱可能是重要的。

Receptors with a seven transmembrane domain structure initiate signal transduction by activation of specific Gα proteins. The aim of this project was to understand the initial step in the signal trnsduction of type 1 angiotensin (AT1) and type 2 angiotensin (AT2) receptors by examining the Gα, protein recognition profile of these receptors. Chimeric Gαs protein constructs, whose receptor binding regions were altered to incorporate amino acids from G proteins from the 4 major families of Gα proteins (Gαq, Gαi, Gα12, Gαs), were cotransfected with AT1 or AT2 receptors in COS cells, then stimulated with angiotensin II (Ang II). Changes in cellular cAMP were assayed on cell lysates by enzyme immuoassay. In the case of the Gαq family, cotransfection of AT1 with Gα11/Gαs, Gα14/Gαs, Gα16/Gαs, elicited significant increases in cAMP after agonist stimulation, whereas cotransfection with Gαs did not cause a significant change. To corroborate these findings, membrane fractions from homogenized vascular smooth muscle cells were stimulated with Ang II in the presence of [35S]GTPγS, then G proteins were immunoprecipitated with antibodies towards Gαq/11, Gα14, and Gαs. Results for these experiments paralleled the results from the chimeric G protein assays. Further examination using chimeric G proteins for Gα12 proteins and Gαi family proteins provided evidence that the AT1 receptor recognizes Gα12, Gαi1/i2, Gαz, Gαo, while both receptors recognized Gαi3. These results suggest Gα protein recognition profiles for both AT1 and AT2 receptors, which may be important for understanding the full spectrum of cellular responses mediated by the hormone Ang II.