title: Functional analysis of human D1 and D5 dopaminergic G protein-coupled receptors: lessons from mutagenesis of a conserved serine residue in the cytosolic end of transmembrane region 6.
abstract: In mammals, dopamine G protein-coupled receptors (GPCR) are segregated into two categories: D1-like (D1R and D5R) and D2-like (D2R(short), D2R(long), D3R, and D4R) subtypes. D1R and D5R are primarily coupled to stimulatory heterotrimeric GTP-binding proteins (Gs/olf) leading to activation of adenylyl cyclase and production of intracellular cAMP. D1R and D5R share high level of amino acid identity in transmembrane (TM) regions. Yet these two GPCR subtypes display distinct ligand binding and G protein coupling properties. In fact, our studies suggest that functional properties reported for constitutively active mutants of GPCRs (e.g., increased basal activity, higher agonist affinity and intrinsic activity) are also observed in cells expressing wild type D5R when compared with wild type D1R. Herein, we describe an experimental method based on mutagenesis and transfection of human embryonic kidney 293 (HEK293) cells to explore the molecular mechanisms regulating ligand affinity, agonist-independent and dependent activity of D1R and D5R. We will demonstrate how to mutate one conserved residue in the cytosolic end of TM6 of D1R (Ser263) and D5R (Ser287) by modifying two or three nucleotides in the cDNA of human D1-like receptors. Genetically modified D1R and D5R cDNAs are prepared using a polymerase chain reaction method, propagated in E. coli, purified and mutations confirmed by DNA sequencing. Receptor expression constructs are transfected into HEK293 cells cultured in vitro at 37°C in 5% CO(2) environment and used in radioligand binding and whole cAMP assays. In this study, we will test the effect of S263A/G/D and S287A/G/D mutations on ligand binding and DA-dependent activation of D1R and D5R.
authors.affiliation: Department of Medicine, Ottawa Hospital Research Institute, University of Ottawa, Ottawa, ON, Canada.
authors.forename: Bianca
authors.initials: B
authors.lastname: Plouffe
authors.forename: Mario
authors.initials: M
authors.lastname: Tiberi
mesh_terms: D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D017124:Conserved Sequence; D000242:Cyclic AMP / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004298:Dopamine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D017447:Receptors, Dopamine D1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050641:Receptors, Dopamine D5 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: D017447:Receptors, Dopamine D1; D050641:Receptors, Dopamine D5; D012694:Serine; D000242:Cyclic AMP; D004262:DNA Restriction Enzymes; D004298:Dopamine