Analysis of G alpha protein recognition profiles of angiotensin II receptors using chimeric G alpha proteins

Receptors with a heptahelical structure initiate signal transduction by int eracting with specific G alpha proteins. The aim of this study was to analy ze the ability of type 1 (AT1) and type 2 (AT2) angiotensin receptors to re cognize the receptor coupling regions of G alpha proteins using our previou sly described technique (Ikezu, T., Okamoto, T., Komatsuzaki, K., Matsui, T ., Martyn, J.A.J., Nishimoto, I., 1996. Negative transactivation of cAMP re sponse element by familial Alzheimer's mutants of APP. EMBO J. 15, 2468-247 5; Komatsuzaki, K., Murayama, Y., Giambarella, U., Ogata, E., Seino, S., Ni shimoto, I., 1996. A novel system that reports the G-proteins linked to a g iven receptor: a study of the type 3 somatostatin receptor. FEBS Lett. 406, 165-170). Chimeric G alphas protein constructs, whose receptor binding reg ions contained sequences from the four major families of G alpha proteins ( G alphaq, G alphai, G alpha 12, G alphas), were cotransfected with AT1 or A T2 receptors in COS cells, then stimulated with angiotensin II (Ang II). Ch anges in cellular cAMP were assayed on cell lysates by enzyme immunoassay. In the case of the G alphaq family, cotransfection of AT1 with G alpha 11/G alphas, G alpha 14/G alphas, G alpha 16/G alphas, elicited significant inc reases in cAMP after agonist stimulation. Confirmatory results were found u sing an independent [S-35]GTP gammaS binding assay. Further examination usi ng chimeric G proteins for G alpha 12 proteins and G alphai family proteins provided evidence that the AT1 receptor can recognize sequences from G alp ha 12, G alpha i1/i2, G alphaz, G alphao, while both receptors interacted w ith G alpha i3. These results provide a G alpha protein recognition databas e for both AT1 and AT2 receptors, which may be important for understanding the full spectrum of cellular responses mediated by the hormone Ang II. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.