More than just a dimer: detection of G protein-coupled receptor oligomers using fluorescent protein reassembly of Ste2p, a yeast pheromone receptor

Cevheroğlu, Orkun
GPCRs are known to form homo- and hetero-dimers and this interaction could have important roles in internalization, maturation, function and/or pharmacology of these receptors. In the first part of the study bimolecular fluorescence complementation (BiFC) using split enhanced green fluorescent protein (EGFP) was used to determine the interaction and cellular location between various Ste2p constructs. Co-expression of two constructs, one with the N-terminus of EGFP inserted into the full-length receptor at the end of the 7th transmembrane domain and the other with the C-terminus of EGFP inserted at the same position, led to the discovery of dimers both at the cell surface and intracellularly as shown by BiFC. Only cell surface dimerization was observed when truncated receptors with the N-terminus of EGFP or the C-terminus of EGFP attached to the 7th transmembrane domain were co-expressed. Co-expression of EGFP-tagged truncated receptors with tagged full-length receptors showed dimers intracellularly and on the plasma membrane indicating that truncated receptor could interact with full-length receptor. Fluorescence as a result of BiFC requires dimer formation, but whether the receptors were also forming higher order aggregates could not be determined by the method used. Using bimolecular fluorescence complementation, we present the evidence that both full length and C-terminally truncated Ste2p traffics to the membrane as a monomer and forms dimers on the cell membrane. C-terminally truncated receptors do not appear to be internalized. In contrast, full-length receptors can internalize as dimers and/or higher oligomers. This study shows that yeast pheromone receptor, Ste2p dimerize on the plasma membrane and are internalized as dimers or oligomers. The BiFC method provided the first evidence of the localization of dimers of truncated and full-length Ste2p. In the second part of the study, BiFC constructs from the first part and new BiFC constructs; truncated receptors with the N-terminus of mCherry or the C-terminus of mCherry attached to the 7th transmembrane domain; as well as, full length or truncated receptors tagged with full length EGFP or mCherry was used to show three and four receptor oligomerization of Ste2p, taking advantage of endocytosis using colocalization and FRET methods.


The yeast Ste2p G protein-coupled receptor dimerizes on the cell plasma membrane
Cevheroglu, Orkun; Kumas, Gozde; Hauser, Melinda; Becker, Jeffrey M.; Son, Çağdaş Devrim (2017-05-01)
Dimerization of G protein-coupled receptors (GPCR) may play an important role in maturation, internalization, signaling and/or pharmacology of these receptors. However, the location where dimerization occurs is still under debate. In our study, variants of Ste2p, a yeast mating pheromone GPCR, were tagged with split EGFP (enhanced green fluorescent protein) fragments inserted between transmembrane domain seven and the C-terminus or appended to the C-terminus. Bimolecular Fluorescence Complementation (BiFC) ...
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Protein-protein interactions (PPIs) have great importance for intracellular signal transduction and sustaining the homeostasis of an organism. Thus, the identification of PPIs is necessary to better understand the downstream signaling functions of the proteins in healthy and pathological conditions. Forster resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful tool for detecting PPIs in living cells. In literature, FRET analysis methods such as donor photobleaching (FLIM), accept...
Citation Formats
O. Cevheroğlu, “More than just a dimer: detection of G protein-coupled receptor oligomers using fluorescent protein reassembly of Ste2p, a yeast pheromone receptor,” Ph.D. - Doctoral Program, Middle East Technical University, 2015.