The constitutively active N111G-AT1 receptor for angiotensin II maintains a high affinity conformation despite being uncoupled from its cognate G protein Gq/11alpha.
Asn111, localized in the third transmembrane domain of the AT1 receptor for angiotensin II, plays a critical role in stabilizing the inactive conformation of the receptor. We evaluated the functional and G protein-coupling properties of mutant AT1 receptors in which Asn111 was substituted with smaller (Ala or Gly) or larger residues (Gln or Trp). All four mutants were expressed at high levels in COS-7 cells and, except for N111W-AT1, recognized 125I-Ang II with high affinities comparable to that of the wild-type AT1 receptor. In phospholipase C assays, the four mutants encompassed the entire spectrum of functional states, ranging from constitutive activity (without agonist) for N111A-AT1 and N111G-AT1 to a significant loss of activity (upon maximal stimulation) for N111Q-AT1 and a major loss of activity for N111W-AT1. In Ca2+ mobilization studies, N111W-AT1 produced a weak Ca2+ transient and, unexpectedly, N111G-AT1 also produced a Ca2+ transient that was much weaker than that of the wild-type AT1. The agonist binding affinity of N111W-AT1 was not modified in the presence of GTPgamma S, suggesting that this receptor is not basally coupled to a G protein. GTPgamma S did not modify the high agonist-binding affinity of N111G-AT1 but abolished the coimmunoprecipitation of Gq/11alpha with this constitutively active mutant receptor. These results are a direct demonstration that the N111G-AT1 receptor maintains a high affinity conformation despite being uncoupled from the G protein Gq/11.
Auger-Messier
Mannix
M
Department of Pharmacology, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4.
Clement
Martin
M
Lanctot
Pascal M
PM
Leclerc
Patrice C
PC
Leduc
Richard
R
Escher
Emanuel
E
Guillemette
Gaetan
G
D000804:Angiotensin II / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D019556:COS Cells; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D016297:Mutagenesis, Site-Directed / Q000502:physiology; D011487:Protein Conformation; D044140:Receptor, Angiotensin, Type 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014662:Vasoconstrictor Agents / Q000378:metabolism / Q000494:pharmacology
D007457:Iodine Radioisotopes; D044140:Receptor, Angiotensin, Type 1; D014662:Vasoconstrictor Agents; D000804:Angiotensin II; D043802:GTP-Binding Protein alpha Subunits, Gq-G11