Showing papers on "GTP-Binding Protein alpha Subunits published in 2016"

Invited review: Activation of G proteins by GTP and the mechanism of Gα-catalyzed GTP hydrolysis.

Abstract: This review addresses the regulatory consequences of the binding of GTP to the alpha subunits (Gα) of heterotrimeric G proteins, the reaction mechanism of GTP hydrolysis catalyzed by Gα and the means by which GTPase activating proteins (GAPs) stimulate the GTPase activity of Gα. The high energy of GTP binding is used to restrain and stabilize the conformation of the Gα switch segments, particularly switch II, to afford stable complementary to the surfaces of Gα effectors, while excluding interaction with Gβγ, the regulatory binding partner of GDP-bound Gα. Upon GTP hydrolysis, the energy of these conformational restraints is dissipated and the two switch segments, particularly switch II, become flexible and are able to adopt a conformation suitable for tight binding to Gβγ. Catalytic site pre-organization presents a significant activation energy barrier to Gα GTPase activity. The glutamine residue near the N-terminus of switch II (Glncat ) must adopt a conformation in which it orients and stabilizes the γ phosphate and the water nucleophile for an in-line attack. The transition state is probably loose with dissociative character; phosphoryl transfer may be concerted. The catalytic arginine in switch I (Argcat ), together with amide hydrogen bonds from the phosphate binding loop, stabilize charge at the β-γ bridge oxygen of the leaving group. GAPs that harbor "regulator of protein signaling" (RGS) domains, or structurally unrelated domains within G protein effectors that function as GAPs, accelerate catalysis by stabilizing the pre-transition state for Gα-catalyzed GTP hydrolysis, primarily by restraining Argcat and Glncat to their catalytic conformations. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 449-462, 2016.

WNT Stimulation Dissociates a Frizzled 4 Inactive-State Complex with Gα12/13.

Abstract: Frizzleds (FZDs) are unconventional G protein-coupled receptors that belong to the class Frizzled. They are bound and activated by the Wingless/Int-1 lipoglycoprotein (WNT) family of secreted lipoglycoproteins. To date, mechanisms of signal initiation and FZD-G protein coupling remain poorly understood. Previously, we showed that FZD(6) assembles withG alpha(i1)/G alpha(q) (but not withG alpha(s), G alpha(o) and G alpha(12/13)), and that these inactive-state complexes are dissociated by WNTs and regulated by the phosphoprotein Dishevelled (DVL). Here, we investigated the inactive-state assembly of heterotrimeric G proteins with FZD(4), a receptor important in retinal vascular development and frequently mutated in Norrie disease or familial exudative vitreoretinopathy. Live-cell imaging experiments using fluorescence recovery after photobleaching show that human FZD(4) assembles-in a DVL-independent manner-with G alpha(12/13) but not representatives of other heterotrimeric G protein subfamilies, such as G alpha(i1), G alpha(o), G alpha(s), andG alpha(q). The FZD(4)-Gprotein complex dissociates upon stimulation with WNT-3A, WNT-5A, WNT-7A, and WNT-10B. In addition, WNT-induced dynamic mass redistribution changes in untransfected and, even more so, in FZD(4) green fluorescent protein-transfected cells depend on G alpha(12/13). Furthermore, expression of FZD(4) and G alpha(12) or G alpha(13) in human embryonic kidney 293 cells induces WNT-dependent membrane recruitment of p115-RHOGEF (RHO guanine nucleotide exchange factor, molecular weight 115 kDa), a direct target of G alpha(12/13) signaling, underlining the functionality of an FZD(4)-G alpha(12/13)-RHO signaling axis. In summary, G alpha(12/13)-mediatedWNT/FZD(4) signaling through p115-RHOGEF offers an intriguing and previously unappreciated mechanistic link of FZD(4) signaling to cytoskeletal rearrangements and RHO signaling with implications for the regulation of angiogenesis during embryonic and tumor development.

Dynamic regulation of neutrophil polarity and migration by the heterotrimeric G protein subunits Gαi-GTP and Gβγ

Abstract: Activation of the Gi family of heterotrimeric guanine nucleotide-binding proteins (G proteins) releases βγ subunits, which are the major transducers of chemotactic G protein-coupled receptor (GPCR)-dependent cell migration. The small molecule 12155 binds directly to Gβγ and activates Gβγ signaling without activating the Gαi subunit in the Gi heterotrimer. We used 12155 to examine the relative roles of Gαi and Gβγ activation in the migration of neutrophils on surfaces coated with the integrin ligand intercellular adhesion molecule-1 (ICAM-1). We found that 12155 suppressed basal migration by inhibiting the polarization of neutrophils and increasing their adhesion to ICAM-1-coated surfaces. GPCR-independent activation of endogenous Gαi and Gβγ with the mastoparan analog Mas7 resulted in normal migration. Furthermore, 12155-treated cells expressing a constitutively active form of Gαi1 became polarized and migrated. The extent and duration of signaling by the second messenger cyclic adenosine monophosphate (cAMP) were enhanced by 12155. Inhibiting the activity of cAMP-dependent protein kinase (PKA) restored the polarity of 12155-treated cells but did not decrease their adhesion to ICAM-1 and failed to restore migration. Together, these data provide evidence for a direct role of activated Gαi in promoting cell polarization through a cAMP-dependent mechanism and in inhibiting adhesion through a cAMP-independent mechanism.

Ric-8A, a G protein chaperone with nucleotide exchange activity induces long-range secondary structure changes in Gα.

Abstract: Cytosolic Ric-8A has guanine nucleotide exchange factor (GEF) activity and is a chaperone for several classes of heterotrimeric G protein α subunits in vertebrates. Using Hydrogen-Deuterium Exchange-Mass Spectrometry (HDX-MS) we show that Ric-8A disrupts the secondary structure of the Gα Ras-like domain that girds the guanine nucleotide-binding site, and destabilizes the interface between the Gαi1 Ras and helical domains, allowing domain separation and nucleotide release. These changes are largely reversed upon binding GTP and dissociation of Ric-8A. HDX-MS identifies a potential Gα interaction site in Ric-8A. Alanine scanning reveals residues crucial for GEF activity within that sequence. HDX confirms that, like G protein-coupled receptors (GPCRs), Ric-8A binds the C-terminus of Gα. In contrast to GPCRs, Ric-8A interacts with Switches I and II of Gα and possibly at the Gα domain interface. These extensive interactions provide both allosteric and direct catalysis of GDP unbinding and release and GTP binding.

Computational Simulation of the Activation Cycle of Gα Subunit in the G Protein Cycle Using an Elastic Network Model.

Abstract: Agonist-activated G protein-coupled receptors (GPCRs) interact with GDP-bound G protein heterotrimers (Gαβγ) promoting GDP/GTP exchange, which results in dissociation of Gα from the receptor and Gβγ. The GTPase activity of Gα hydrolyzes GTP to GDP, and the GDP-bound Gα interacts with Gβγ, forming a GDP-bound G protein heterotrimer. The G protein cycle is allosterically modulated by conformational changes of the Gα subunit. Although biochemical and biophysical methods have elucidated the structure and dynamics of Gα, the precise conformational mechanisms underlying the G protein cycle are not fully understood yet. Simulation methods could help to provide additional details to gain further insight into G protein signal transduction mechanisms. In this study, using the available X-ray crystal structures of Gα, we simulated the entire G protein cycle and described not only the steric features of the Gα structure, but also conformational changes at each step. Each reference structure in the G protein cycle was modeled as an elastic network model and subjected to normal mode analysis. Our simulation data suggests that activated receptors trigger conformational changes of the Gα subunit that are thermodynamically favorable for opening of the nucleotide-binding pocket and GDP release. Furthermore, the effects of GTP binding and hydrolysis on mobility changes of the C and N termini and switch regions are elucidated. In summary, our simulation results enabled us to provide detailed descriptions of the structural and dynamic features of the G protein cycle.