Systematic identification of mutations that constitutively activate the angiotensin II type 1A receptor by screening a randomly mutated cDNA library with an original pharmacological bioassay

The constitutive activation of G-protein-coupled receptors is a major new a pproach to investigating their physiopathology and pharmacology. A large nu mber of spontaneous and site-directed mutations resulting in constitutive a ctivity have been identified, but systematic mapping of the amino acids inv olved for a given receptor would be extremely useful for complete elucidati on of the molecular mechanisms underlying its activation. We carried out su ch mapping for the angiotensin II type 1A (AT(1A)) receptor by screening a randomly mutated cDNA library after expressing the mutated clones in eukary otic cells. To test the AT1A mutants generated, we developed an original, s pecific, and highly sensitive assay based on the properties of CGP42112A. T his classical AT(2) agonist is a weak partial agonist of the wild-type AT1A receptor and becomes a full agonist for constitutively active AT1A mutants , as shown experimentally and in allostery-based theoretical models. Activa tion of the mutated receptors by CGP42112A was monitored by using the biolu minescent protein aequorin, a very sensitive and specific sensor of intrace llular calcium mobilization. The screening of 4,800 clones, providing an ex haustive coverage of all of the mutations generated, led to the identificat ion of 16 mutations in sequences encoding the transmembrane domains that we re responsible for high sensitivity to CGP42112A. The constitutive activity was confirmed by agonist-independent production of inositol phosphates. wh ich showed that at least half of the clones had significantly increased bas al activity. These data demonstrate that this new type of approach is very efficient for the systematic identification of constitutively active mutant s of G-protein-coupled receptors.