Doctoral Dissertations

Date of Award

12-2004

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Jeffrey M. Becker

Committee Members

Elizabeth E. Howell, Engin Serpersu, John W. Koontz, Salil K. Niyogi

Abstract

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 ofiodinatable 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 cross-linked 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 cross-linked 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.

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