Temperature-induced Ligand Contact Point Variations of the hAT1 Receptor and of the Constitutively Active Mutant N111G-hAT1
G-protein coupled receptors (GPCR) are crucial for intracellular signalling and primary targets for pharmacological intervention. Among typical peptidergic GPCRs, the human Angiotensin II (AngII) type 1 receptor, the hAT1 receptor is an important pharmacological target and has been extensively scrutinized as a prototypical receptor for many years with methods such as photoaffinity labeling. In the present study, a variant of the Methionine Proximity Assay (MPA) (1) was applied to hAT1 to investigate the effects of temperature on labeling and contact points of a neutral agonist on the receptor. Selected methionine mutants, located in transmembrane domain 6 (TMDVI); F249M, W253M and H256M of the hAT1 and its constitutively active mutant (CAM) N111G-hAT1, were photolabeled with 125I-[Sar1, Bpa8] AngII at specific and controlled temperatures ranging from −15°C to 37°C. These methionines were observed as contact points in previous studies (2, 4).
From a preliminary experiment,...
KeywordsPhotoaffinity Label Label Frequency Mutant Series Human Angiotensin Inactive Receptor
We would like to thank CIHR and CHSFQ for our funding, B. Holleran, R. Hamel, E. Wieczerzak and M.-R. Lefebvre for their help and technical support.
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