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Novel Mechanisms of G Protein-Coupled Receptors
Date of Award
Doctor of Philosophy (PhD)
More than 800 human G protein-coupled receptors (GPCRs) comprise a superfamily of membrane-bound proteins that respond to extracellular stimuli and activate intracellular signaling by catalyzing the exchange of GDP for GTP on linked heterotrimeric G proteins. The work described in this thesis evaluates previously unknown mechanisms by which GPCRs regulate intracellular signaling and establishes methods to identify novel GPCR-stimulated protein interactions and modifications.
First, Frizzled receptors were investigated for their ability to interact with G proteins. Frizzled receptors are known to signal through non-G protein mediated mechanisms to control differentiation and developmental processes but have characteristic features of GPCRs, including seven transmembrane domains. Yeast genetic approaches employed in this thesis demonstrated that Frizzled receptors do interact with G-alpha-i, G-alpha-q, and G-alpha-s proteins. However, the activity level of Frizzled-mediated G protein signaling was much lower than that of a typical GPCR and was highest when coupled to G-alpha-s. Additional studies identified an interaction between Frizzled and G-alpha-s in Drosophila. Together, these data point to an important role for Frizzled as a nontraditional GPCR that preferentially couples to G-alpha-s heterotrimeric G proteins.
Second, the roles of conserved extracellular cysteine residues in the N-terminus and third extracellular loop (EC3) were examined for novel roles in regulating receptor function. These cysteines are found in approximately 10% of the rhodopsin-like family of GPCRs and in nearly all chemokine receptors. The studies in this thesis focus on the human nicotinic acid receptor, GPR109A, which contains an additional pair of cysteine residues in the N-terminus and EC2. To assess for functional coupling of the extracellular cysteines, mammalian cells that stably overexpress wild-type or cysteine-mutated GPR109A were assayed for adenylate cyclase inhibition in response to ligand stimulation. These studies showed that extracellular cysteine residues are important for normal GPR109A function and that the amino terminal cysteines play non-redundant roles. Intriguingly, removal of the N-terminus/EC2 cysteine pair that is unique to GPR109A restored wild-type function to GPR109A, suggesting that the N-terminus/EC3 cysteine pair found in chemokine receptors plays the major role in positively regulating GPR109A function. This finding suggests that other potential disulfide pairs might be responsible for downregulating GPR109A-mediated signaling under different redox conditions.
Finally, the thesis outlines proteomics techniques that utilize mass spectrometry to identify post-translational modifications as well as receptor binding partners of nicotinic acid-treated GPR109A. Preliminary work to map the extracellular disulfide bonds of GPR109A by analyzing sulfhydryl labeled receptors by mass spectrometry is also described.
Chair and Committee
Thomas J. Baranski
Kendall J. Blumer, Michael R. Bruchas, William A. Frazier, N. Gautam, Karen L. O'Malley
Nichols, Andrea, "Novel Mechanisms of G Protein-Coupled Receptors" (2013). Arts & Sciences Electronic Theses and Dissertations. 91.