A constitutively active GPCR retains its G protein specificity and the ability to form dimers.
G protein-coupled receptors (GPCRs) are cell surface proteins which help to regulate the physiology of all the major organ systems within higher eukaryotes. They are stimulated by multiple ligands and activate a range of effector molecules to bring about changes in cell behaviour. The use of constitutively active mutants (CAMs) of GPCRs has enabled a better understanding of receptor activation as CAMs exhibit ligand-independent signalling negating the use of ligands. Here we introduce the fission yeast Schizosaccharomyces pombe as a host for producing CAMs, by describing the isolation and characterization of constitutive mutants of the P-factor receptor (Mam2). One mutant Mam2[P261L] contained a single-amino-acid substitution (Pro261 to Leu) within a region of high homology in GPCRs. Substitution of this proline leads to an 18-fold increase in ligand-independent signalling. We utilized Mam2[P261L] to investigate CAM activity by demonstrating that Mam2[P261L] is efficiently trafficked to the cell surface where it can form fully functional oligomeric complexes with the native receptor. Mam2[P261L] also retains the G protein specificity (RG-profile) of the native receptor and only induces constitutive signalling in the same G proteins. Finally, evidence is provided to indicate that CAM activity results from a reduction in the kinetics of G protein binding. This is the first time that S. pombe has been utilized for isolating and characterizing CAMs and the techniques employed will complement the current systems available for studying these important receptors.
Ladds
Graham
G
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK. Graham.ladds@warwick.ac.uk
Davis
Kevin
K
Das
Anamika
A
Davey
John
J
D000595:Amino Acid Sequence; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D019281:Dimerization; D005656:Fungal Proteins; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation; D010675:Pheromones / Q000378:metabolism*; D021381:Protein Transport; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism*
D005656:Fungal Proteins; D008024:Ligands; D010675:Pheromones; D043562:Receptors, G-Protein-Coupled; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors; C085847:map2 protein, S pombe