2014
DOI: 10.1002/bit.25278
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Construction of a yeast‐based signaling biosensor for human angiotensin II type 1 receptor via functional coupling between Asn295‐mutated receptor and Gpa1/Gi3 chimeric Gα

Abstract: Angiotensin II (Ang II) type 1 receptor (AGTR1) is a G-protein-coupled receptor (GPCR). Its natural ligand, Ang II, is an important effector molecule controlling blood pressure and volume in the cardiovascular system, and is consequently involved in various diseases such as hypertension and heart failure. Thus, the signaling mediator, AGTR1, is a significant molecular target in medicinal and therapeutic fields. Yeast is a useful organism for sensing GPCR signaling because it provides a simplified version of th… Show more

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Cited by 11 publications
(14 citation statements)
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“…NTSR1 signaling assays were performed according to previously described procedures. [16,33] Briefly, the yeast strains transformed with the NTSR1 expression plasmids were grown in SD medium (supplemented with amino acids and/or nucleic-acid base as needed) at 30 C overnight; the cells then were inoculated into 5 mL of the respective fresh SD medium to give an initial OD 600 ¼ 0.03. The cells were incubated at 30 C on a rotary shaker at 150 rpm for up to 18 h, then harvested, washed, and resuspended in distilled water to yield an OD 600 ¼ 10.…”
Section: Ntsr1 Signaling Assay With Exogenously Added Ligandsmentioning
confidence: 99%
“…NTSR1 signaling assays were performed according to previously described procedures. [16,33] Briefly, the yeast strains transformed with the NTSR1 expression plasmids were grown in SD medium (supplemented with amino acids and/or nucleic-acid base as needed) at 30 C overnight; the cells then were inoculated into 5 mL of the respective fresh SD medium to give an initial OD 600 ¼ 0.03. The cells were incubated at 30 C on a rotary shaker at 150 rpm for up to 18 h, then harvested, washed, and resuspended in distilled water to yield an OD 600 ¼ 10.…”
Section: Ntsr1 Signaling Assay With Exogenously Added Ligandsmentioning
confidence: 99%
“…Specifically, a previously described EGFP reporter yeast strain was re‐engineered by replacing a pair of EGFP reporter genes with ZsGreen genes, while also replacing the endogenous GPA1 locus with a chimeric gene coding for a yeast Gpa1‐human Gα i3 protein (Gpa1/Gα i3 transplant; G i3 tp). The resulting yeast strain showed more than 30‐fold increased fluorescence than the previous EGFP reporter strain, and the improved version was used to detect agonist‐promoted signaling by several human GPCRs whose natural ligands were short peptides, such as somatostatin, neurotensin, and angiotensin (Nakamura et al, , ).…”
Section: Introductionmentioning
confidence: 99%
“…Flow cytometry measurements of green and far‐red fluorescence were performed according to a previously described procedure (Nakamura et al, ,, ). In brief, fluorescent cells were detected using a BD FACSCanto II flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ) equipped with both a 488‐nm blue laser and a 633‐nm red laser; the data were analyzed using BD FACSDiva software (v5.0; Becton, Dickinson and Company).…”
Section: Methodsmentioning
confidence: 99%