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

Bianca Plouffe, Mario Tiberi

Research output: Chapter in Book/Report/Conference proceedingChapter

5 Citations (Scopus)

Abstract

In mammals, dopamine G protein-coupled receptors (GPCR) are segregated into two categories: D1-like (D1R and D5R) and D2-like (D2Rshort, D2Rlong, 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, puri fied and mutations con firmed 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.

Original languageEnglish
Title of host publicationDopamine
Subtitle of host publicationMethods and Protocols
PublisherHumana Press Inc.,U.S.
Pages141-180
Number of pages40
ISBN (Print)9781627032506
DOIs
Publication statusPublished - 01 Jan 2013
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume964
ISSN (Print)1064-3745

Keywords

  • CAMP
  • D1-like receptors
  • Dopamine
  • GPCR
  • HEK293 cells
  • Ligand binding
  • Mutagenesis
  • Third intracellular loop
  • TM6

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Medicine(all)

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