title: One motif to bind them: A small-XXX-small motif affects transmembrane domain 1 oligomerization, function, localization, and cross-talk between two yeast GPCRs.
abstract: G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors in mammals and facilitate a range of physiological responses triggered by a variety of ligands. GPCRs were thought to function as monomers, however it is now accepted that GPCR homo- and hetero-oligomers also exist and influence receptor properties. The Schizosaccharomyces pombe GPCR Mam2 is a pheromone-sensing receptor involved in mating and has previously been shown to form oligomers in vivo. The first transmembrane domain (TMD) of Mam2 contains a small-XXX-small motif, overrepresented in membrane proteins and well-known for promoting helix-helix interactions. An ortholog of Mam2 in Saccharomyces cerevisiae, Ste2, contains an analogous small-XXX-small motif which has been shown to contribute to receptor homo-oligomerization, localization and function. Here we have used experimental and computational techniques to characterize the role of the small-XXX-small motif in function and assembly of Mam2 for the first time. We find that disruption of the motif via mutagenesis leads to reduction of Mam2 TMD1 homo-oligomerization and pheromone-responsive cellular signaling of the full-length protein. It also impairs correct targeting to the plasma membrane. Mutation of the analogous motif in Ste2 yielded similar results, suggesting a conserved mechanism for assembly. Using co-expression of the two fungal receptors in conjunction with computational models, we demonstrate a functional change in G protein specificity and propose that this is brought about through hetero-dimeric interactions of Mam2 with Ste2 via the complementary small-XXX-small motifs. This highlights the potential of these motifs to affect a range of properties that can be investigated in other GPCRs.
authors.affiliation: Department of Genetics, Evolution and Environment, University College London, WC1E 6BT London, UK.
authors.forename: Antonia
authors.initials: A
authors.lastname: Lock
authors.affiliation: Division of Biomedical Cell Biology, Warwick Medical School, Coventry, CV4 7AL, UK.
authors.forename: Rachel
authors.initials: R
authors.lastname: Forfar
authors.affiliation: Division of Biomedical Cell Biology, Warwick Medical School, Coventry, CV4 7AL, UK.
authors.forename: Cathryn
authors.initials: C
authors.lastname: Weston
authors.affiliation: Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK.
authors.forename: Leo
authors.initials: L
authors.lastname: Bowsher
authors.affiliation: School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK.
authors.forename: Graham J G
authors.initials: GJ
authors.lastname: Upton
authors.affiliation: School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Reynolds
authors.affiliation: Division of Biomedical Cell Biology, Warwick Medical School, Coventry, CV4 7AL, UK.. Electronic address: graham.ladds@warwick.ac.uk.
authors.forename: Graham
authors.initials: G
authors.lastname: Ladds
authors.affiliation: Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK. Electronic address: ann.dixon@warwick.ac.uk.
authors.forename: Ann M
authors.initials: AM
authors.lastname: Dixon
keywords: Cross-talk; GPCR; Oligomerization; Signaling; TOXCAT; Trafficking