Temperature-induced Ligand Contact Point Variations of the hAT1 Receptor and of the Constitutively Active Mutant N111G-hAT1

摘要

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 ~(125)I-[Sar, Bpa] Angll at specific and controlled temperatures ranging from -15°C to 37°C. These methionines were observed as contact points in previous studies (2,4).