Molecular cloning of a ferret angiotensin II AT_1 receptor reveals the importance of position 163 for Losartan binding

摘要

A complementary DNA for the angiotensin II (AngII) type 1 (AT_1) receptor from Mustela putorius furo (ferret) was isolated from a ferret atria cDNA library. The cDNA encodes a protein (fAT_1) of 359 amino acids having high homologies (93-99%) to other mammalian AT-1 receptor counterparts. When fAT_1 was expressed in COS-7 cells and photoaffinity labeled with the photoactive analogue ~(125)I-[Sar~1, Bpa~8]AngII, a protein of 100 kDa was detected by autoradiography. The formation of this complex was specific since it was abolished in the presence of the AT_1 non-peptidic antagonist L-158, 809. Functional analysis indicated that the fAT_1 receptor efficiently coupled to phospholipase C as demonstrated by an increase in inositol phosphate production following stimulation with AngII. Binding studies revealed that the fAT_1 receptor had a high affinity for the peptide antagonist [Sar~1, Ile~8]AngII (K_d of 508 ± 1.4 nM) but a low affinity for the AT-1 selective non-peptidic antagonist DuP 753 (K_d of 91 ± 15.6 nM). Interestingly, when we substituted Thr~(163) with an Ala residue, which occupies this position in many mammalian AT_1 receptors, we restored the high affinity of this receptor for Dup 753 (11.7 ± 5.13 nM). These results suggest that position 163 of the AT_1 receptor does not contribute to the overall binding of peptidic ligands but that certain non-peptidic antagonists such as Dup 753 are clearly dependent on this position for efficient binding.