ABSTRACT
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 G(q)/PLC signaling in transfected cells using AngII and the signaling selective agonist [Sar(1),Ile(4),Ile(8)] AngII (SII). SII stimulated MAPK without inositol trisphosphate (IP(3)) 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-Tyr(292), Phe(293), and Thr(287). At least three distinct activated states were revealed: 1) an AngII-stabilized state linked to G(q)/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 IP(3) turnover yet remained capable of AngII-induced MAPK activation. SII failed to stimulate MAPK in Y292F-transfected cells. Thus, Tyr(292) 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 IP(3) response to SII, indicating that Phe(293) may be involved in constraining the receptor to its inactive state. Mutations of Thr(287) abolished all SII-induced signaling without affecting any AngII responses. Thr(287) therefore represents a key residue for a SII-stabilized activated state. Taken together, the data identified a novel structural requirement (Thr(287)) for the SII-stabilized activated state and redefined the mechanistic roles for Tyr(292) and Phe(293).
Subject(s)
Enzyme Activation/physiology , Extracellular Signal-Regulated MAP Kinases/physiology , GTP-Binding Proteins/physiology , Receptor, Angiotensin, Type 1/chemistry , Animals , COS Cells , Chlorocebus aethiops , Conserved Sequence , Extracellular Signal-Regulated MAP Kinases/metabolism , GTP-Binding Proteins/metabolism , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Models, Biological , Mutation , Point Mutation , Protein Structure, Quaternary , Protein Structure, Tertiary , Rats , Receptor, Angiotensin, Type 1/agonists , Receptor, Angiotensin, Type 1/genetics , Receptor, Angiotensin, Type 1/metabolism , TransfectionABSTRACT
Examination of the Rattus norvegicus genome reveals differences in the melanocortin 3 receptor (MC3R) compared with the published sequence (accession X70667). To clarify these differences, we used RT-PCR to clone MC3R from Sprague Dawley rats. These efforts revealed a sequence for the rat MC3R consistent with that predicted by the rat genome, but different from the published receptor by three amino acids, all of which were located in the predicted second transmembrane domain (TM2). Analysis of these residues revealed that TM2 of the rat MC3R is more homologous with other species than previously considered. The presently described sequence maps onto chromosome 3 of the rat genome, which shows highly conserved synteny with the mouse chromosome 2 and the human chromosome 20. Transient expression revealed high affinity binding of [125I]-NDP-MSH and a concentration-dependent cAMP response to the synthetic agonist MTII. These data both clarify the sequence of the MC3R and demonstrate the great utility of genomic information recently made available.
