Structural insights into the molecular basis of heterotrimeric G-protein signal transduction

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  • March 21, 2019
Creator
  • Johnston, Christopher Alan
    • Affiliation: School of Medicine, Department of Pharmacology
Abstract
  • Heterotrimeric (αβγ) G-proteins serve as molecular switches in response to diverse extracellular signals. G-protein activation occurs through the guanine nucleotide exchange factor (GEF) activity of surface-bound G-protein coupled receptors (GPCRs), which catalyze the exchange of GDP for GTP within the Gα subunit. GTP-bound Gα and freed Gβγ subunits regulate a wide range of downstream effector molecules participating in cellular physiology. G-protein deactivation occurs through the intrinsic hydrolysis of GTP to GDP by Gα and reassociation with Gβγ. The Regulators of G-protein Signaling (RGS) family of proteins, which act as GTPase Accelerating Proteins (GAPs), can dramatically accelerate this deactivation rate. Despite extensive investigation, several aspects regarding the molecular mechanisms of G-protein signal transduction remain undefined and/or incompletely understood. First, the precise mechanisms of receptor coupling and subsequent G-protein activation have eluded definition. How do heterotrimeric G-proteins selectively couple to receptors, and how do activated receptors elicit conformational changes in the G-protein required for GDP release? Second, the spatiotemporal dynamics of G-protein activation have not been well characterized. How do the timing and spatial aspects of G-protein activation contribute to proper signal transduction within the cell? Third, the exact biological role of the RGS 'box' and its associated GAP activity have not been thoroughly examined to date. Do RGS proteins confer additional signaling mechanisms beyond the well-characterized GAP activity, and how does GTPase activity per se affect the biological output in response to a stimulus input? Finally, emerging, yet not fully appreciated, roles for heterotrimeric Gproteins in several model organisms continue to expand our knowledge of the biological function of these critical signaling proteins. What are the molecular mechanisms involved in G-protein regulated signal transduction in these systems and how do they relate to G-protein signaling in humans? It is therefore the aim of this dissertation to further develop our understanding of the molecular mechanisms governing these and other aspects of heterotrimeric G-protein signaling. A combination of biochemical, structural, and biological techniques has been employed throughout to achieve these goals. The data described herein shed new light on several heretofore unanswered questions regarding the molecular basis of heterotrimeric G-protein signaling. Collectively, our results highlight novel aspects of G-protein signaling dynamics. Firstly, we have determined the molecular basis for two bona fide receptor/G-protein contact sites: one between the third intracellular loop of the D2-dopamine receptor and the α4/β6 region of Gα and another between the C-terminal loop of the PTH1 receptor and the WD1 and WD7 repeats of Gβ. Together with results from a unique, phage display peptide, KB- 752, our work suggests a potential mechanism through which receptors activate heterotrimeric G-proteins: direct receptor/Gα contacts are used to manipulate the β6/α5 loop and destabilize GDP binding, while simultaneously direct receptor/Gβ contacts are used to alter the inhibitory conformation of the β3/α2 loop allowing for a feasible GDP exit route. Secondly, we have developed a novel activation-dependent Gα binding peptide, KB- 1753, capable of detecting the GTP-dependent conformational changes induced within Gα. The Gα/KB-1753 crystal structure represents the first ‘effector’-bound structure for Gαi1 and shares molecular determinant features common to known Gα/effector pairs. The ability of KB-1753 to selectively interact with Gαi family members despite high sequence conservation among all Gα families within its binding site provides insight into the effector recognition determinants for Gα subunits. We have also developed KB-1753-based biosensors for the detection of Gα activation and its spatiotemporal nature in vivo. Thirdly, using a combination of biochemistry, crystallography, and genetics, we provide novel insights into the regulation of GTP hydrolysis within Gα and the physiological role of RGS protein GAP activity. We demonstrate that the β3/α2 loop is a critical determinant of switch II conformational dynamics and serves as a basal regulator of intrinsic GTP hydrolysis by modulating the ability to adopt the transition state conformation. Using yeast as a model organism, we show that RGS protein function in vivo likely exceeds the simplistic view that these proteins serve as mere negative regulators of signaling via associated GAP activity. Finally, work in both A. thaliana and C. elegans model organisms helps further define the regulatory roles for G-protein signaling in critical cellular functions. A GTP hydrolysislimited nucleotide cycle of AtGPA1 is shown to regulate sugar sensing in A. thaliana, while a complex interplay between GOA-1 and GPA1 regulates cell division in C. elegans.
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  • In Copyright
Advisor
  • Siderovski, David P.
Degree granting institution
  • University of North Carolina at Chapel Hill
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  • Open access
Date uploaded
  • October 19, 2010

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