Analysis of transmembrane domains 1 and 4 of the human angiotensin II AT1 receptor by cysteine-scanning mutagenesis.

The Journal of Biological Chemistry
Li Ping YanRichard Leduc

Abstract

The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the AT(1) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT(1) receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. Here, we investigated the role of the first and fourth transmembrane domains (TMDs) in the formation of the binding pocket of the human AT(1) receptor using the substituted-cysteine accessibility method. Each residue within the Phe-28((1.32))-Ile-53((1.57)) fragment of TMD1 and Leu-143((4.40))-Phe-170((4.67)) fragment of TMD4 was mutated, one at a time, to a cysteine. The resulting mutant receptors were expressed in COS-7 cells, which were subsequently treated with the charged sulfhydryl-specific alkylating agent methanethiosulfonate ethylammonium (MTSEA). This treatment led to a significant reduction in the binding affinity of TMD1 mutants M30C((1.34))-AT(1) and T33C((1.37))-AT(1) and TMD4 mutant V169C((4.66))-AT(1). Although this reduction in binding of the TMD1 mutants was maintained when examined in a constitutively active receptor (N111G-AT(1)) backgr...Continue Reading

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Citations

Dec 12, 2012·The Journal of Biological Chemistry·Jérôme CabanaPierre Lavigne
Mar 7, 2014·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Leo T O LeeBilly K C Chow
Aug 1, 2018·Scientific Reports·Elena Tutunea-FatanStephen S G Ferguson
Sep 24, 2013·Journal of Chemical Information and Modeling·Minos-Timotheos MatsoukasPanagiotis Zoumpoulakis

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