|Budget Amount *help
¥1,500,000 (Direct Cost : ¥1,500,000)
Fiscal Year 1999 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1998 : ¥800,000 (Direct Cost : ¥800,000)
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.