Identification of Structural Determinants for G Protein-Independent Activation of Mitogen-Activated Protein Kinases in the Seventh Transmembrane Domain of the Angiotensin II Type 1 Receptor

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Although the intrareceptor mechanisms whereby the angiotensin II (AngII) type 1 receptor activates phospholipase C (PLC) have been extensively investigated, analogous studies of signaling through mitogen-activated protein kinases (MAPK) have been lacking. We investigated MAPK activation and traditional Gq/PLC signaling in transfected cells using AngII and the signaling selective agonist [Sar1,Ile4,Ile8] AngII (SII). SII stimulated MAPK without inositol trisphosphate (IP3) production and thereby stabilizes an activated receptor state linked to G protein-independent MAPK signaling. Using receptor mutagenesis, we focused on the seventh transmembrane domain and identified three key residues—Tyr292, Phe293, and Thr287. At least three distinct activated states were revealed: 1) an AngII-stabilized state linked to Gq/PLC signaling, 2) an AngII-stabilized state connected to G protein-independent MAPK activation, and 3) a SII-stabilized state associated with G protein-independent MAPK signaling. The mutant Y292F failed to exhibit AngII-induced IP3 turnover yet remained capable of AngII-induced MAPK activation. SII failed to stimulate MAPK in Y292F-transfected cells. Thus, Tyr292 is a key epitope for activated states 1 and 3 but not required for activated state 2. Although the F293L mutant retained normal AngII responses, it also showed an IP3 response to SII, indicating that Phe293 may be involved in constraining the receptor to its inactive state. Mutations of Thr287 abolished all SII-induced signaling without affecting any AngII responses. Thr287 therefore represents a key residue for a SII-stabilized activated state. Taken together, the data identified a novel structural requirement (Thr287) for the SII-stabilized activated state and redefined the mechanistic roles for Tyr292 and Phe293.

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