An angiotensin II type 1 receptor activation switch patch revealed through evolutionary trace analysis.
Seven transmembrane (7TM) or G protein-coupled receptors constitute a large superfamily of cell surface receptors sharing a structural motif of seven transmembrane spanning alpha helices. Their activation mechanism most likely involves concerted movements of the transmembrane helices, but remains to be completely resolved. Evolutionary Trace (ET) analysis is a computational method, which identifies clusters of functionally important residues by integrating information on evolutionary important residue variations with receptor structure. Combined with known mutational data, ET predicted a patch of residues in the cytoplasmic parts of TM2, TM3, and TM6 to form an activation switch that is common to all family A 7TM receptors. We tested this hypothesis in the rat Angiotensin II (Ang II) type 1a (AT1a) receptor. The receptor has important roles in the cardiovascular system, but has also frequently been applied as a model for 7TM receptor activation and signaling. Six mutations: F66A, L67R, L70R, L119R, D125A, and I245F were targeted to the putative switch and assayed for changes in activation state by their ligand binding, signaling, and trafficking properties. All but one receptor mutant (that was not expressed well) displayed phenotypes associated with changed activation state, such as increased agonist affinity or basal activity, promiscuous activation, or constitutive internalization highlighting the importance of testing different signaling pathways. We conclude that this evolutionary important patch mediates interactions important for maintaining the inactive state. More broadly, these observations in the AT1 receptor are consistent with computational predictions of a generic role for this patch in 7TM receptor activation.
Laboratory for Molecular Cardiology, The Danish National Research Foundation Centre for Cardiac Arrhythmia, The Heart Centre, Copenhagen University Hospital, Rigshospitalet, Juliane Mariesvej 20, section 9312, DK-2100 Copenhagen, Denmark.
Marie Mi
MM
Bonde
Rong
R
Yao
Jian-Nong
JN
Ma
Srinivasan
S
Madabushi
Stig
S
Haunsø
Ethan S
ES
Burstein
Jennifer L
JL
Whistler
Søren P
SP
Sheikh
Olivier
O
Lichtarge
Jakob Lerche
JL
Hansen
D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D005075:Biological Evolution; D003593:Cytoplasm / Q000378:metabolism; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D044140:Receptor, Angiotensin, Type 1 / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics
D044140:Receptor, Angiotensin, Type 1; D043562:Receptors, G-Protein-Coupled; D000804:Angiotensin II