title: 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 the complicated machinery used by mammalian cells for signal transduction. Although yeast cells can successfully transmit a signal through a variety of human GPCRs expressed in the cell membrane, there have been no reports of the functional activation of AGTR1-mediated signaling in yeast cells. In the present study, we introduced a single mutation into human AGTR1 and used yeast-human chimeric Gα to exert the functional activation of AGTR1 in yeast cells. The engineered yeast cells expressing AGTR1 mutated at Asn295 and the chimeric Gα successfully transmitted the signal inside the yeast cells in response to Ang II peptide and its analogs (Ang III and Ang IV peptides) added to the assay medium. Further, we demonstrated that the autocrine Ang II peptide and its analog, produced and secreted by the engineered yeast cells, could by themselves promote AGTR1-mediated signaling. This means that screening for agonistic peptides with various sequences from a self-produced genetic library would be a viable strategy. Thus, the constructed yeast biosensor, integrating an Asn295-mutated AGTR1 receptor, will be valuable in the design of drugs to treat AGTR1-related diseases.
authors.affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan.
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Nakamura
authors.forename: Jun
authors.initials: J
authors.lastname: Ishii
authors.forename: Akihiko
authors.initials: A
authors.lastname: Kondo
mesh_terms: D000804:Angiotensin II / Q000032:analysis*; D015374:Biosensing Techniques / Q000379:methods*; D006801:Humans; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D017354:Point Mutation; D044140:Receptor, Angiotensin, Type 1 / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D015398:Signal Transduction
chemical_list: C544065:AGTR1 protein, human; D050505:Mutant Proteins; D044140:Receptor, Angiotensin, Type 1; D011994:Recombinant Proteins; D000804:Angiotensin II
keywords: G-protein signaling; G-protein-coupled receptor; Saccharomyces cerevisiae; angiotensin II type 1 receptor; green fluorescent protein; peptide screening