The fifth transmembrane domain of angiotensin II type 1 receptor participates in the formation of the ligand binding pocket and undergoes a counterclockwise rotation upon receptor activation. Export

@article{citeulike:5998089, abstract = {The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the AT1 receptor, which belongs to the G protein-coupled receptors (GPCR) superfamily. Like other GPCRs, the AT1 receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. The role of the fifth transmembrane domain (TMD5) was investigated using the substituted-cysteine accessibility method. All the residues within Thr-190 to Leu-217 region were mutated one at a time to cysteine and, after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of L197C-AT1, N200C-AT1, I201C-AT1, G203C-AT1 and F204C-AT1 mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD5 reporter cysteines engineered in a constitutively active N111G-AT1 receptor background. Indeed, mutant I201C-N111G-AT1 became more sensitive to MTSEA, whereas mutant G203C-N111G-AT1 lost some sensitivity. Our results suggest that constitutive activation of AT1 receptor causes an apparent counterclockwise rotation of TMD5 as viewed from the extracellular side.}, author = {Domazet, Ivana and Martin, Stephane S. and Holleran, Brian J. and Morin, Marie-Eve E. and Lacasse, Patrick and Lavigne, Pierre and Escher, Emanuel and Leduc, Richard and Guillemette, Gaetan}, citeulike-article-id = {5998089}, citeulike-linkout-0 = {http://dx.doi.org/10.1074/jbc.M109.051839}, citeulike-linkout-1 = {http://view.ncbi.nlm.nih.gov/pubmed/19773549}, citeulike-linkout-2 = {http://www.hubmed.org/display.cgi?uids=19773549}, day = {22}, doi = {10.1074/jbc.M109.051839}, issn = {1083-351X}, journal = {The Journal of biological chemistry}, keywords = {activation, angiotensin, cys\_scanning, tm5}, month = {September}, posted-at = {2009-10-23 16:17:52}, priority = {2}, title = {The fifth transmembrane domain of angiotensin II type 1 receptor participates in the formation of the ligand binding pocket and undergoes a counterclockwise rotation upon receptor activation.}, url = {http://dx.doi.org/10.1074/jbc.M109.051839}, year = {2009} }

TY - JOUR ID - citeulike:5998089 L3 - citeulike-article-id:5998089 N2 - The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the AT1 receptor, which belongs to the G protein-coupled receptors (GPCR) superfamily. Like other GPCRs, the AT1 receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. The role of the fifth transmembrane domain (TMD5) was investigated using the substituted-cysteine accessibility method. All the residues within Thr-190 to Leu-217 region were mutated one at a time to cysteine and, after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of L197C-AT1, N200C-AT1, I201C-AT1, G203C-AT1 and F204C-AT1 mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD5 reporter cysteines engineered in a constitutively active N111G-AT1 receptor background. Indeed, mutant I201C-N111G-AT1 became more sensitive to MTSEA, whereas mutant G203C-N111G-AT1 lost some sensitivity. Our results suggest that constitutive activation of AT1 receptor causes an apparent counterclockwise rotation of TMD5 as viewed from the extracellular side. JF - The Journal of biological chemistry SN - 1083-351X TI - The fifth transmembrane domain of angiotensin II type 1 receptor participates in the formation of the ligand binding pocket and undergoes a counterclockwise rotation upon receptor activation. KW - activation KW - angiotensin KW - cys_scanning KW - tm5 AU - Domazet, Ivana AU - Martin, Stephane AU - Holleran, Brian AU - Morin, Marie-Eve AU - Lacasse, Patrick AU - Lavigne, Pierre AU - Escher, Emanuel AU - Leduc, Richard AU - Guillemette, Gaetan PY - 2009/09/22/ UR - http://dx.doi.org/10.1074/jbc.M109.051839 ER -