Bower, Kiowa San (2010) Chemical-scale studies of the 5-HT3 and D2 dopamine receptors. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechTHESIS:03142010-163350106
During synaptic transmission in the central nervous system, neuroreceptors transduce a chemical signal into an electrical signal, a process that is mediated by both ligand-gated ion channels (LGICs) and G-protein coupled receptors (GPCRs). The work in this thesis examines structure-function relationships within these receptors, with a focus on elucidating the mechanism of molecular recognition during ligand binding. We utilize conventional and unnatural amino acid mutagenesis, structural derivatives of agonists, and homology models to identify specific interactions and the role of binding site residues in ligand binding and receptor activation. The technique of unnatural amino acid mutagenesis allows us to study these processes in greater detail than would otherwise be possible, even at the scale of a chemical bond. Chapter 2 covers structure-function investigations of a ligand-gated ion channel, the 5-HT_3 receptor, with a goal of understanding agonist binding and receptor activation. The project examines residues in close proximity to the ligand-binding site and focuses on polar interactions with hydrophilic residues. We identify 5-fluorotryptamine (5-FT) as a partial agonist of the 5-HT_3 receptors and show that size and electronegativity are important at the 5’ position for efficient channel opening. Our investigation of the compound 1-OT revealed it to be an agonist of equal potency to the native agonist (5-HT), demonstrating that the indolic proton of serotonin is not essential to its activation of the receptor. A study focusing on loop A residues led us to refine our homology model and propose that Glu129 faces into the binding pocket, where, through its ability to hydrogen bond, it plays a critical role in ligand binding. Further studies of binding site residues identified an ionic interaction that likely participates in the conformational changes associated with receptor gating and characterized several other residues that play critical roles in receptor activation. Finally, we compare and contrast the behaviors of two structurally distinct agonist classes, 5-HT and its related structures, and m-chlorophenylbiguanide (mCPBG) and identify several residues that play critical roles in modulating agonist binding and gating in response to these agonists. Chapter 3 describes a study examining the binding site and the mechanism of agonist activation of a GPCR, the D2 dopamine receptor. A number of aromatic amino acids thought to be near the agonist binding site were evaluated. Incorporation of a series of fluorinated tryptophan derivatives at a conserved tryptophan of the D2 receptor establishes a cation-π interaction between the agonist dopamine and this residue (W6.48), suggesting a reorientation of W6.48 on agonist binding, consistent with proposed “rotamer switch” models. Finally, chapter 4 describes a project that seeks to extend the nonsense suppression methodology to include mammalian expression systems. Progress is made developing techniques for efficient transfection of cells in culture.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Subject Keywords:||G-protein-coupled receptors; ligand-gated ion channels; unnatural amino acids; 5-HT3; D2 dopamine; 5-fluorotryptamine|
|Degree Grantor:||California Institute of Technology|
|Major Option:||Biochemistry and Molecular Biophysics|
|Thesis Availability:||Public (worldwide access)|
|Defense Date:||13 January 2010|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Kiowa Bower|
|Deposited On:||04 Jun 2010 17:31|
|Last Modified:||22 Aug 2016 21:18|
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