Akademik Veri Yönetim Sistemi
Tez Türü: Doktora
Tezin Yürütüldüğü Kurum: University of Tennessee, Knoxville, Fen Edebiyat Fakültesi, Biyokimya, Hücresel ve Moleküler Biyoloji, Amerika Birleşik Devletleri
Tez Danışmanı: Jeffrey M. Becker
Tezin Onay Tarihi: 2004
Tezin Dili: İngilizce
Açık Arşiv Koleksiyonu: AVESİS Açık Erişim Koleksiyonu
Özet:
G protein-coupled receptors (GPCRs) are a class of integral membrane receptor
proteins that are characterized by a signature seven-transmembrane (7TM) configuration.
These receptors comprise a large and diverse gene family found in fungi, plants, and the
animal kingdom. Recent studies with GPCRs have begun to elucidate their importance in
many physiological processes, thus various human diseases are associated with GPCR
pathology. Although the overall 3D structure of these receptors carry similar features,
binding of an extraordinarily diverse array of ligands trigger many different biological
pathways.
The α-factor receptor (Ste2p) of Saccharomyces cerevisiae belongs to the GPCR
family. Upon the α-factor binding to Ste2p, a signal is transduced via an associated
guanine-nucleotide binding protein initiating a cascade of events that leads to the mating
of haploid yeast cells. As only two GPCRs and two G proteins are encoded in the S.
cerevisiae genome, this yeast presents a relatively simple system to study GPCR signal
transduction in comparison to mammalian cells that possess hundreds of GPCRs and tens
of G proteins. Part I of this dissertation is an overview of GPCRs in general with specific
emphasis on the peptide pheromone α-factor and its receptorSte2p.
Part II of this dissertation details the design and characterization of a number of
iodinatable α-factor pheromone analogs containing the photo-cross-linkable 4-benzoyl-Lphenylalanine
(Bpa) group. One of these analogs [Bpa1, Y3, R7, Nle12, F13] was
radioiodinated for detection and used as a probe for cross-linking studies with Ste2p.
Chemical (with CNBr & BNPS-skatole) and enzymatic (with Trypsin) cleavage of the
receptor/analog complex after the cross-linking was examined to determine the
interaction between the α-factor probe and a fragment of the receptor. Data from these
digestions indicated that the position one of the α-factor interacts with residues 251 to
294 in the receptor.
Similarly Part III of this dissertation describes the design and synthesis of five
photoactivatable α-factor analogs that carry Bpa at positions one, three, five, eight, or
thirteen. All of these analogs were biotinylated at the ε-amine of the Lys7 for detection
and purification purposes. The biological activity (growth arrest assay) and binding
affinities of all analogs for Ste2p were determined. Two of the analogs tested, Bpa1 and
Bpa5, showed three- to four-fold lower affinity compared to α-factor, whereas Bpa3 and
Bpa13 had seven- to twelve-fold lower affinities, respectively. Bpa8 competed poorly with
[3H]α-factor for Ste2p. All of the analogs tested had detectable halos in the growth arrest
assay indicating that these analogs are α-factor agonists. Cross-linking studies
demonstrated that [Bpa1]α-factor, [Bpa3]α-factor, [Bpa5]α-factor and [Bpa13]α-factor
were cross-linked to Ste2p; the biotin tag on the pheromone was detected by a
NeutrAvidin-HRP conjugate on Western blots. Digestion of Bpa1, Bpa3, and Bpa13 crosslinked
receptors with chemical and enzymatic reagents suggested that the N-terminus of
the pheromone interacts with a binding domain consisting of residues from the
extracellular ends of TM5, TM6, and TM7 and portions of EL2 and EL3 close to these
TMs. Additionally it was concluded that there is a direct interaction between the position
13 side chain and a region of Ste2p (F55-R58) at the extracellular end of TM1. Parts II
and III of this dissertation indicate that Bpa-containing α-factor probes are useful in
determining contacts between α-factor and Ste2p and initiating mapping of the ligand
binding site of the GPCR for its peptide ligand.
This dissertation (Part IV) also presents the application of different purification
methods and the use of two mass spectrometry instruments for identification of ligandreceptor
interactions at the molecular level. Results presented in this part showed that
although a single step purification was enough for western blot analyses of the crosslinked
receptor fragments, at least a two-step purification and enrichment of the
biotinylated peptide fragments were necessary for mass spectrometric studies. MALDITOF
experiments showed that the affinity purification of the biotinylated fragments by
monomeric avidin beads was successful. Data obtained from CNBr fragments of Bpa1
cross-linked membranes were in agreement with the previous results discussed in Parts II
and III of this dissertation suggesting the cross-linking between position one of α-factor
and a region of Ste2p covering residues 251 to 294. This part also illustrated that the
analyses of the MS/MS data from the cross-linked fragments were more complex than the
fragmentation data obtained from biotinylated α-factor; the presence of multiple charge
states of fragment ions and unusual fragmentation of branched peptides indicated the
necessity of using an instrument with higher resolution. In addition, analyses of the
MS/MS data with a customized algorithm would be required to deconvolute the sequence
of the cross-linked fragment(s) to identify the cross-linked residue(s) on Ste2p.
The final part of this dissertation reviews the overall conclusions and discussion.
This part also contains suggestions for future experiments that could help identification of
contact points between Ste2p and its peptide ligand α-factor. Additional studies on this
GPCR system employing high-resolution mass spectral characterization of fragments
should allow identification of residue-to-residue interactions between the analogs used in
this study and Ste2p. Such information will aid the mapping of the ligand-binding site of
the pheromone receptor and has the potential to provide key insights into peptide ligand
mediated activation of GPCRs. This and similar studies may ultimately lead to the
discovery of how peptide ligands initiate signal transduction through GPCRs.