Akademik Veri Yönetim Sistemi
Tezin Türü: Yüksek Lisans
Tezin Yürütüldüğü Kurum: Orta Doğu Teknik Üniversitesi, Fen Bilimleri Enstitüsü, Türkiye
Tezin Onay Tarihi: 2019
Tezin Dili: İngilizce
Öğrenci: ÖZGE ATAY
Asıl Danışman (Eş Danışmanlı Tezler İçin): Çağdaş Devrim Son
Eş Danışman: Salih Özçubukçu
Özet:
The classical GPCR signaling pathway, where a heterotrimeric G protein-GPCR
interaction is sufficient to transmit the signal to effector proteins has been
replaced by a heteromeric G protein-GPCR homo- or hetero-dimer interaction
model over the past two decades. These studies demonstrate that GPCRs that
interact with each other couple with a heteromeric G protein. In recent years,
evidence suggests that dimer of GPCR dimers is required for some complex signal transductions.
In these studies, it was proposed that this heteromeric tetramer formed by the
dimerization of the dimers brought two G proteins close enough to each other
for protein-protein interaction. It is not clear if GPCR tetramerization is
required for G-protein dimerization or dimerization can occur independent of
the GPCRs. On the other hand, studies on small G-proteins (Ras family), which
are structural homologs of G alpha subunits of heteromeric G-proteins, shows
that dimerization can be independent of the receptors and necessary for
various signaling pathways.
Within the scope of
this study, Gα protein homodimerizations were qualitatively and quantitatively
investigated in live cells using Bimolecular Fluorescent Complementation Assay
(BiFC) and Förster resonance energy transfer (FRET) method. To achieve this,
the Gαi1 protein gene was labeled from various positions, including (G60-Y61, L91-K92 and A121-E122), with Enhance Green
Fluorescent Protein (EGFP) which was derived from Aequorea victoria and mCherry fluorescent protein
which is a monomeric derivative of DsRed.
In addition, the Gαi1 gene was labeled with split EGFP parts which are
N-terminus EGFP and C-terminus EGFP for the Bimolecular Fluorescence
Complementation Assay. All labeled proteins were co-transfected into Mus
musculus Neuroblastoma-2a (N2a) cells and interactions were imaged using spinning
disc confocal microscope and analyzed.
The findings of this study could
help us understand the molecular mechanisms required for Gα dimerization and
the dynamics of these proteins. Also, GPCR interactions with various effectors
during complex signal transductions and the requirement of these receptors
during Gα dimerizations can be studied with the techniques optimized in this
study.