William M. Oldham

Heterotrimeric G protein activation by G-protein-coupled receptors

Heterotrimeric G proteins have a crucial role as molecular switches in signal transduction pathways mediated by G-protein-coupled receptors. Extracellular stimuli activate these receptors, which then catalyse GTP–GDP exchange on the G protein α-subunit. The complex series of interactions and conformational changes that connect agonist binding to G protein activation raise various interesting questions about the structure, biomechanics, kinetics and specificity of signal transduction across the plasma membrane.

Structural basis of function in heterotrimeric G proteins

Heterotrimeric guanine-nucleotide-binding proteins (G proteins) act as molecular switches in signaling pathways by coupling the activation of heptahelical receptors at the cell surface to intracellular responses. In the resting state, the G-protein alpha subunit (Galpha) binds GDP and Gbetagamma. Receptors activate G proteins by catalyzing GTP for GDP exchange on Galpha, leading to a structural change in the Galpha(GTP) and Gbetagamma subunits that allows the activation of a variety of downstream effector proteins. The G protein returns to the resting conformation following GTP hydrolysis and subunit re-association. As the G-protein cycle progresses, the Galpha subunit traverses through a series of conformational changes. Crystallographic studies of G proteins in many of these conformations have provided substantial insight into the structures of these proteins, the GTP-induced structural changes in Galpha, how these changes may lead to subunit dissociation and allow Galpha and Gbetagamma to activate effector proteins, as well as the mechanism of GTP hydrolysis. However, relatively little is known about the receptor-G protein complex and how this interaction leads to GDP release from Galpha. This article reviews the structural determinants of the function of heterotrimeric G proteins in mammalian systems at each point in the G-protein cycle with special emphasis on the mechanism of receptor-mediated G-protein activation. The receptor-G protein complex has proven to be a difficult target for crystallography, and several biophysical and computational approaches are discussed that complement the currently available structural information to improve models of this interaction. Additionally, these approaches enable the study of G-protein dynamics in solution, which is becoming an increasingly appreciated component of all aspects of G-protein signaling.

Mechanism of the receptor-catalyzed activation of heterotrimeric G proteins

Heptahelical receptors activate intracellular signaling pathways by catalyzing GTP for GDP exchange on the heterotrimeric G protein α subunit (Gα). Despite the crucial role of this process in cell signaling, little is known about the mechanism of G protein activation. Here we explore the structural basis for receptor-mediated GDP release using electron paramagnetic resonance spectroscopy. Binding to the activated receptor (R*) causes an apparent rigid-body movement of the α5 helix of Gα that would perturb GDP binding at the β6-α5 loop. This movement was not observed when a flexible loop was inserted between the α5 helix and the R*-binding C terminus, which uncouples R* binding from nucleotide exchange, suggesting that this movement is necessary for GDP release. These data provide the first direct observation of R*-mediated conformational changes in G proteins and define the structural basis for GDP release from Gα.

Hypoxia-Mediated Increases in l-2-hydroxyglutarate Coordinate the Metabolic Response to Reductive Stress

Metabolic adaptation to hypoxia is critical for survival in metazoan species for which reason they have developed cellular mechanisms for mitigating its adverse consequences. Here, we have identified L-2-hydroxyglutarate (L2HG) as a universal adaptive determinant of the hypoxia response. L2HG is a metabolite of unknown function produced by the reduction of mitochondrial 2-oxoglutarate by malate dehydrogenase. L2HG accumulates in response to increases in 2-oxoglutarate, which occur as a result of tricarboxylic acid cycle dysfunction and increased mitochondrial reducing potential. These changes are closely coupled to cellular redox homeostasis, as increased cellular L2HG inhibits electron transport and glycolysis to offset the adverse consequences of mitochondrial reductive stress induced by hypoxia. Thus, L2HG couples mitochondrial and cytoplasmic energy metabolism in a model of cellular redox regulation.

How do receptors activate G proteins

Heterotrimeric G proteins couple the activation of heptahelical receptors at the cell surface to the intracellular signaling cascades that mediate the physiological responses to extracellular stimuli. G proteins are molecular switches that are activated by receptor-catalyzed GTP for GDP exchange on the G protein alpha subunit, which is the rate-limiting step in the activation of all downstream signaling. Despite the important biological role of the receptor-G protein interaction, relatively little is known about the structure of the complex and how it leads to nucleotide exchange. This chapter will describe what is known about receptor and G protein structure and outline a strategy for assembling the current data into improved models for the receptor-G protein complex that will hopefully answer the question as to how receptors flip the G protein switch.

Vascular stiffness mechanoactivates YAP/TAZ-dependent glutaminolysis to drive pulmonary hypertension

Dysregulation of vascular stiffness and cellular metabolism occurs early in pulmonary hypertension (PH). However, the mechanisms by which biophysical properties of the vascular extracellular matrix (ECM) relate to metabolic processes important in PH remain undefined. In this work, we examined cultured pulmonary vascular cells and various types of PH-diseased lung tissue and determined that ECM stiffening resulted in mechanoactivation of the transcriptional coactivators YAP and TAZ (WWTR1). YAP/TAZ activation modulated metabolic enzymes, including glutaminase (GLS1), to coordinate glutaminolysis and glycolysis. Glutaminolysis, an anaplerotic pathway, replenished aspartate for anabolic biosynthesis, which was critical for sustaining proliferation and migration within stiff ECM. In vitro, GLS1 inhibition blocked aspartate production and reprogrammed cellular proliferation pathways, while application of aspartate restored proliferation. In the monocrotaline rat model of PH, pharmacologic modulation of pulmonary vascular stiffness and YAP-dependent mechanotransduction altered glutaminolysis, pulmonary vascular proliferation, and manifestations of PH. Additionally, pharmacologic targeting of GLS1 in this model ameliorated disease progression. Notably, evaluation of simian immunodeficiency virus-infected nonhuman primates and HIV-infected subjects revealed a correlation between YAP/TAZ-GLS activation and PH. These results indicate that ECM stiffening sustains vascular cell growth and migration through YAP/TAZ-dependent glutaminolysis and anaplerosis, and thereby link mechanical stimuli to dysregulated vascular metabolism. Furthermore, this study identifies potential metabolic drug targets for therapeutic development in PH.

Mapping allosteric connections from the receptor to the nucleotide-binding pocket of heterotrimeric G proteins

Heterotrimeric G proteins function as molecular relays that mediate signal transduction from heptahelical receptors in the cell membrane to intracellular effector proteins. Crystallographic studies have demonstrated that guanine nucleotide exchange on the Gα subunit causes specific conformational changes in three key “switch” regions of the protein, which regulate binding to Gβγ subunits, receptors, and effector proteins. In the present study, nitroxide side chains were introduced at sites within the switch I region of Gαi to explore the structure and dynamics of this region throughout the G protein cycle. EPR spectra obtained for each of the Gα(GDP), Gα(GDP)βγ heterotrimer and Gα(GTPγS) conformations are consistent with the local environment observed in the corresponding crystal structures. Binding of the heterotrimer to activated rhodopsin to form the nucleotide-free (empty) complex, for which there is no crystal structure, causes prominent changes relative to the heterotrimer in the structure of switch I and contiguous sequences. The data identify a putative pathway of allosteric changes triggered by receptor binding and, together with previously published data, suggest elements of a mechanism for receptor-catalyzed nucleotide exchange.

Structural and dynamical changes in an α-subunit of a heterotrimeric G protein along the activation pathway

The Gα subunits of heterotrimeric G proteins (Gαβγ) mediate signal transduction via activation by receptors and subsequent interaction with downstream effectors. Crystal structures indicate that conformational changes in “switch” sequences of Gα, controlled by the identity of the bound nucleotide (GDP and GTP), modulate binding affinities to the Gβγ subunits, receptor, and effector proteins. To investigate the solution structure and dynamics of Gαi1 through the G protein cycle, nitroxide side chains (R1) were introduced at sites in switch II and at a site in helix α4, a putative effector binding region. In the inactive Gαi1(GDP) state, the EPR spectra are compatible with conformational polymorphism in switch II. Upon complex formation with Gβγ, motions of R1 are highly constrained, reflecting direct contact interactions at the Gαi1–Gβ interface; remarkably, the presence of R1 at the sites investigated does not substantially affect the binding affinity. Complex formation between the heterotrimer and activated rhodopsin leads to a dramatic change in R1 motion at residue 217 in the receptor-binding α2/β4 loop and smaller allosteric changes at the Gαi1–Gβγ interface distant from the receptor binding surface. Upon addition of GTPγS, the activated Gαi1(GTP) subunit dissociates from the complex, and switch II is transformed to a unique conformation similar to that in crystal structures but with a flexible backbone. A previously unreported activation-dependent change in α4, distant from the interaction surface, supports a role for this helix in effector binding.

Gbetagamma activates GSK3 to promote LRP6-mediated beta-catenin transcriptional activity.

A Xenopus reconstitution system reveals that the G protein Gβγ subunit contributes to β-catenin stabilization. Gβγ for β-Catenin Stability G proteins influence the Wnt–β-catenin pathway, which regulates various developmental processes; aberrant activity is associated with some cancers. The ligand Wnt interacts with a receptor complex that includes the seven-transmembrane protein Frizzled and the single-transmembrane protein LRP6 to activate the transcriptional regulatory activity of β-catenin. By screening the activity of purified G protein subunits in a Xenopus egg extract system, Jernigan et al. found that, in addition to a subset of Gα subunits, the Gβγ subunit also stabilized β-catenin. Various biochemical analyses, including analysis of transfected mammalian cells and in vitro assays, along with the use of a Gβγ-selective inhibitor, suggested that Gβγ recruited the kinase GSK3 to the membrane. After membrane recruitment, GSK3 phosphorylated LRP6, which then inhibited the β-catenin degradation complex, allowing β-catenin to translocate to the nucleus and activate transcription. Additionally, the Gβγ inhibitor prevented axis duplication of Xenopus embryos under conditions of excess LRP6 activity, thus verifying in vivo a role for Gβγ in this pathway. The Gβγ inhibitor failed to block Wnt-mediated activation of β-catenin, which suggests that a receptor other than Frizzled may activate the G protein that contributes to β-catenin signaling. Evidence from Drosophila and cultured cell studies supports a role for heterotrimeric guanosine triphosphate–binding proteins (G proteins) in Wnt signaling. Wnt inhibits the degradation of the transcriptional regulator β-catenin. We screened the α and βγ subunits of major families of G proteins in a Xenopus egg extract system that reconstitutes β-catenin degradation. We found that Gαo, Gαq, Gαi2, and Gβγ inhibited β-catenin degradation. Gβ1γ2 promoted the phosphorylation and activation of the Wnt co-receptor low-density lipoprotein receptor–related protein 6 (LRP6) by recruiting glycogen synthase kinase 3 (GSK3) to the membrane and enhancing its kinase activity. In both a reporter gene assay and an in vivo assay, c-βARK (C-terminal domain of β-adrenergic receptor kinase), an inhibitor of Gβγ, blocked LRP6 activity. Several components of the Wnt–β-catenin pathway formed a complex: Gβ1γ2, LRP6, GSK3, axin, and dishevelled. We propose that free Gβγ and Gα subunits, released from activated G proteins, act cooperatively to inhibit β-catenin degradation and activate β-catenin–mediated transcription.

Direct Modulation of Phospholipase D Activity by Gβγ

Phospholipase D-mediated hydrolysis of phosphatidylcholine is stimulated by protein kinase C and the monomeric G proteins Arf, RhoA, Cdc42, and Rac1, resulting in complex regulation of this enzyme. Using purified proteins, we have identified a novel inhibitor of phospholipase D activity, Gβγ subunits of heterotrimeric G proteins. G protein-coupled receptor activation alters affinity between Gα and Gβγ subunits, allowing subsequent interaction with distinct effectors. Gβ1γ1 inhibited phospholipase D1 and phospholipase D2 activity, and both Gβ1γ1 and Gβ1γ2 inhibited stimulated phospholipase D1 activity in a dosedependent manner in reconstitution assays. Reconstitution assays suggest this interaction occurs through the amino terminus of phospholipase D, because Gβ1γ1 is unable to inhibit an amino-terminally truncated phospholipase D construct, PLD1.d311, which like full-length phospholipase D isoforms, requires phosphatidylinositol-4,5-bisphosphate for activity. Furthermore, a truncated protein consisting of the amino-terminal region of phospholipase D containing the phox/pleckstrin homology domains was found to interact with Gβ1γ1, unlike the PLD1.d311 recombinant protein, which lacks this domain. In vivo, expressed recombinant Gβ1γ2 was also found to inhibit phospholipase D activity under basal and stimulated conditions in MDA-MB-231 cells, which natively express both phospholipase D1 and phospholipase D2. These data demonstrate that Gβγ directly regulates phospholipase D activity in vitro and suggest a novel mechanism to negatively regulate phospholipase D signaling in vivo.