Date of Award

2015

Embargo Period

8-1-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cell and Molecular Pharmacology and Experimental Therapeutics

College

College of Graduate Studies

First Advisor

Joe B. Blumer

Second Advisor

Steve M. Lanier

Third Advisor

John D. Hildebrandt

Fourth Advisor

Carl Atkinson

Fifth Advisor

Lauren E. Ball

Abstract

Activators of G-protein Signaling (AGS) proteins modulate G-protein signaling in diverse and unexpected ways and have functional roles in numerous physiological systems. The functions of AGS proteins in the immune system, where Gαi-coupled chemokine receptors are predominantly involved in dynamic signaling events, are poorly understood. The Group II AGS proteins AGS3 and AGS4 express multiple G-protein regulatory (GPR) motifs, each of which dock GDP-bound Gαi/o/t subunits and effectively compete with Gβγ for binding. This unique ability positions these proteins to modulate downstream signaling of Gαi and Gβγ, thus promoting signal diversity from seven-transmembrane receptors (7TMR). However, regulatory mechanisms and functional roles for the Gαi2–GPR module in leukocytes are poorly understood. Using a bioluminescence resonance energy transfer (BRET) platform, we demonstrated chemokine regulation of Gαi2–GPR modules that were receptor-proximal. Generation of fusion proteins with Gαi physically tethered to the 7TMR revealed that regulation of Gαi2–GPR was independent of endogenous G-protein cycling subsequent to receptor activation, suggesting that Gαi-GPR couples to 7TMRs analogous to Gαβγ heterotrimer. Additional modes of regulation for AGS4 were also investigated including identification of alternative binding proteins ARID1b and eEF1d, suggesting potential modulatory functions for AGS4 in transcription and protein translation, and phosphorylation of AGS4-Y108 by JAK2 and Src, which regulates the Gαi-AGS4 interaction. Furthermore, regulation of Gαi2–GPR by chemokine receptors and expression of AGS3 and AGS4 in immune cells and tissues suggested functional roles of these proteins in the immune system. Investigating chemoattractant signal processing in primary leukocytes from wild-type, AGS3-null and AGS4-null mice demonstrated 25-40% decreased migration with corresponding reduction in ERK1/2 activation of null-animals. The importance of the Gαi–GPR interaction in chemokine signaling provides a novel platform for development of pathway targeted small molecules, identified in preliminary screening for modulators of the Gαi–GPR interaction. These studies have broad implications for G-protein signal processing and Gαi-GPR complexes in immune function.

Rights

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