Date

2015

Author

Cevheroğlu, Orkun

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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.

Subject Keywords

Confocal fluorescence microscopy., G proteins., Pheromones., Oligomers., Dimers., Yeast.

URI

http://etd.lib.metu.edu.tr/upload/12619189/index.pdf
https://hdl.handle.net/11511/25234

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Graduate School of Natural and Applied Sciences, Thesis