Analysis of Galpha protein recognition profiles of angiotensin II receptors using chimeric Galpha proteins

Receptors with a heptahelical structure initiate signal transduction by interacting with specific Galpha proteins. The aim of this study was to analyze the ability of type 1 (AT1) and type 2 (AT2) angiotensin receptors to recognize the receptor coupling regions of Galpha proteins using our previously described technique (Ikezu, T., Okamoto, T., Komatsuzaki, K., Matsui, T., Martyn, J.A.J., Nishimoto, I., 1996. Negative transactivation of cAMP response element by familial Alzheimer's mutants of APP. EMBO J. 15, 2468-2475; Komatsuzaki, K., Murayama, Y., Giambarella, U., Ogata, E., Seino, S., Nishimoto, I., 1996. A novel system that reports the G-proteins linked to a given receptor: a study of the type 3 somatostatin receptor. FEBS Lett. 406, 165-170). Chimeric Galphas protein constructs, whose receptor binding regions contained sequences from the four major families of Galpha proteins (Galphaq, Galphai, Galpha12, Galphas), 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 immunoassay. In the case of the Galphaq family, cotransfection of AT1 with Galpha11/Galphas, Galpha14/Galphas, Galpha16/Galphas, elicited significant increases in cAMP after agonist stimulation. Confirmatory results were found using an independent [35S]GTPgammaS binding assay. Further examination using chimeric G proteins for Galpha12 proteins and Galphai family proteins provided evidence that the AT1 receptor can recognize sequences from Galpha12, Galphai1/i2, Galphaz, Galphao, while both receptors interacted with Galphai3. These results provide a Galpha protein recognition database for both AT1 and AT2 receptors, which may be important for understanding the full spectrum of cellular responses mediated by the hormone Ang II.