title: Antidiabetic sulfonylureas and cAMP cooperatively activate Epac2A.
abstract: Sulfonylureas are widely used drugs for treating insulin deficiency in patients with type 2 diabetes. Sulfonylureas bind to the regulatory subunit of the pancreatic β cell potassium channel that controls insulin secretion. Sulfonylureas also bind to and activate Epac2A, a member of the Epac family of cyclic adenosine monophosphate (cAMP)-binding proteins that promote insulin secretion through activation of the Ras-like guanosine triphosphatase Rap1. Using molecular docking simulation, we identified amino acid residues in one of two cyclic nucleotide-binding domains, cNBD-A, in Epac2A predicted to mediate the interaction with sulfonylureas. We confirmed the importance of the identified residues by site-directed mutagenesis and analysis of the response of the mutants to sulfonylureas using two assays: changes in fluorescence resonance energy transfer (FRET) of an Epac2A-FRET biosensor and direct sulfonylurea-binding experiments. These residues were also required for the sulfonylurea-dependent Rap1 activation by Epac2A. Binding of sulfonylureas to Epac2A depended on the concentration of cAMP and the structures of the drugs. Sulfonylureas and cAMP cooperatively activated Epac2A through binding to cNBD-A and cNBD-B, respectively. Our data suggest that sulfonylureas stabilize Epac2A in its open, active state and provide insight for the development of drugs that target Epac2A.
authors.affiliation: 1Division of Molecular and Metabolic Medicine, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.
authors.forename: Toshimasa
authors.initials: T
authors.lastname: Takahashi
authors.forename: Tadao
authors.initials: T
authors.lastname: Shibasaki
authors.forename: Harumi
authors.initials: H
authors.lastname: Takahashi
authors.forename: Kenji
authors.initials: K
authors.lastname: Sugawara
authors.forename: Aika
authors.initials: A
authors.lastname: Ono
authors.forename: Naoko
authors.initials: N
authors.lastname: Inoue
authors.forename: Toshio
authors.initials: T
authors.lastname: Furuya
authors.forename: Susumu
authors.initials: S
authors.lastname: Seino
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics; D019556:COS Cells; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007004:Hypoglycemic Agents / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D013453:Sulfonylurea Compounds / Q000737:chemistry / Q000378:metabolism*
chemical_list: D020662:Guanine Nucleotide Exchange Factors; D007004:Hypoglycemic Agents; C486707:Rapgef4 protein, mouse; D013453:Sulfonylurea Compounds; D006639:Histidine; D000242:Cyclic AMP