Small GTP-Binding Proteins

doi:10.1016/S0074-7696(08)61861-6

International Review of Cytology

Volume 133, 1992, Pages 187-230

https://doi.org/10.1016/S0074-7696(08)61861-6 Get rights and content

Publisher Summary

This chapter highlights small G proteins. It also reviews their possible functions and modes of action in comparison with those of ras p21. Three groups of G proteins have both GDP/GTP-binding and GTPase activities. The first group of G proteins includes G proteins involved in protein synthesis. The second group of G proteins comprises a superfamily of the G_s, G_i, G_o, G_q, and transducin families. G proteins of the third group are monomeric and show molecular weight values between 20,000 and 36,000. These G proteins are called “small G proteins” and are composed of more than 40 members. Small G proteins play important roles in regulating various cell functions. The G proteins involved in protein synthesis and the heterotrimeric G proteins serving as transducers for membrane receptors have two interconvertible forms, GDP-bound inactive and GTP-bound active forms. The GDP-bound inactive form is converted to the GTP-bound active form by a GDP/GTP exchange reaction. All of the small G proteins have the consensus amino acid sequences responsible for GDP/GTP-binding and GTPase activities. Small G proteins also possess unique C-terminal sequences.

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Accumulating experimental evidence emphasizes the importance of nitric oxide (NO)-mediated posttranslational modifications of signaling proteins. The small GTP-binding proteins Ras and Rac1 are molecular switches exchanging GDP for GTP and converting external signals in response to a variety of stimuli. Ras and Rac1 play an important role in cell proliferation, cell differentiation, and cell migration. Rac1 is directly involved in the reorganization and changes in the cytoskeleton during cell motility. In endothelial cells, NO stimulates the Ras-ERK1/2 MAP kinases signaling pathway and is involved in the interaction between Ras and the phosphatidyl-inositol-3 Kinase (PI3K) signaling pathway and cell migration. In this chapter we discuss the participation of Ras, Rac1, and PI3K in the NO-mediated endothelial cell migration. The implications for tumor cell angiogenesis are also discussed.
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Astrocytes modulate and support neuronal and synapse function via numerous mechanisms that often rely on diffusion of signalling molecules, ions or metabolites through extracellular space. As a consequence, the spatial arrangement and the distance between astrocyte processes and neuronal structures are of functional importance. Likewise, changes of astrocyte structure will affect the ability of astrocytes to interact with neurons. In contrast to neurons, where rapid morphology changes are critically involved in many aspects of physiological brain function, a role of astrocyte restructuring in brain physiology is only beginning to emerge. In neurons, small GTPases of the Rho family are powerful initiators and modulators of structural changes. Less is known about the functional significance of these signalling molecules in astrocytes. Here, we review recent experimental evidence for the role of RhoA, Cdc42 and Rac1 in controlling dynamic astrocyte morphology as well as experimental tools and analytical approaches for studying astrocyte morphology changes.
Occupation of nucleotide in the binding pocket is critical to the stability of Rab11A
2016, Protein Expression and Purification
Citation Excerpt :
Each GTPase has conserved amino acid sequences of the enzymatic active site that are responsible for specific interactions with GDP and GTP molecules [3]. The activity of GTPase is mainly regulated by bound guanine nucleotides, with the GTP- and GDP bound form indicating the active and inactive state, respectively [4–6]. These forms are interconvertible via GDP/GTP exchange and GTPase reactions, and the transition between the active and inactive form is accompanied by the conformational changes in two highly mobile regions, the switch I and switch II domains [7].
The Ras superfamily of small G proteins is a family of guanosine triphosphatases (GTPases) and each GTPase has conserved amino acid sequences in the enzymatic active site that are responsible for specific interactions with GDP and GTP molecules. Rab GTPases, which belong to the Ras superfamily, are key regulators of intracellular vesicle trafficking via the recruitment of effector molecules. Here, we purified wild type, active mutant and inactive mutant of Rab11A. In this process, we found that the inactive mutant (Rab11A S25N) had low stability compared with wild type and other mutants. Further analysis revealed that the stability of Rab11A S25N is dependent on the occupation of GDP in the nucleotide binding pocket of the protein. We found that the stability of Rab11A S25N is affected by the presence of GDP, not other nucleotides, and is independent of pH or salt in FPLC buffer. Our results provide a better understanding of how GTPase can be stable under in vitro conditions without effector proteins and how proper substrate/cofactor coordination is crucial to the stability of Rab11A. Successful purification and proposed purification methods will provide a valuable guide for investigation of other small GTPase proteins.
Phosphorylation of Rab5a protein by protein kinase Cε Is crucial for T-cell migration
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Rab GTPases control membrane traffic and receptor-mediated endocytosis. Within this context, Rab5a plays an important role in the spatial regulation of intracellular transport and signal transduction processes. Here, we report a previously uncharacterized role for Rab5a in the regulation of T-cell motility. We show that Rab5a physically associates with protein kinase Cϵ (PKCϵ) in migrating T-cells. After stimulation of T-cells through the integrin LFA-1 or the chemokine receptor CXCR4, Rab5a is phosphorylated on an N-terminal Thr-7 site by PKCϵ. Both Rab5a and PKCϵ dynamically interact at the centrosomal region of migrating cells, and PKCϵ-mediated phosphorylation on Thr-7 regulates Rab5a trafficking to the cell leading edge. Furthermore, we demonstrate that Rab5a Thr-7 phosphorylation is functionally necessary for Rac1 activation, actin rearrangement, and T-cell motility. We present a novel mechanism by which a PKCϵ-Rab5a-Rac1 axis regulates cytoskeleton remodeling and T-cell migration, both of which are central for the adaptive immune response.
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Geranylgeranyl diphosphate synthase (GGPPS) catalyzes the condensation of the non-allylic diphosphate, isopentenyl diphosphate (IPP; C₅), with allylic diphosphates to generate the C₂₀ prenyl chain (GGPP) used for protein prenylation and diterpenoid biosynthesis. Here, we cloned the cDNA of a GGPPS from the spruce budworm, Choristoneura fumiferana, and characterized the corresponding recombinant protein (rCfGGPPS). As shown for other type-III GGPPSs, rCfGGPPS preferred farnesyl diphosphate (FPP; C₁₅) over other allylic substrates for coupling with IPP. Unexpectedly, rCfGGPPS displayed inhibition by its FPP substrate at low IPP concentration, suggesting the existence of a mechanism that may regulate intracellular FPP pools. rCfGGPPS was also inhibited by its product, GGPP, in a competitive manner with respect to FPP, as reported for human and bovine brain GGPPSs. A homology model of CfGGPPS was prepared and compared to human and yeast GGPPSs. Consistent with its enzymological properties, CfGGPPS displayed a larger active site cavity that can accommodate the binding of FPP and GGPP in the region normally occupied by IPP and the allylic isoprenoid tail, and the binding of GGPP in an alternate orientation seen for GGPP binding to the human protein. To begin exploring the role of CfGGPPS in protein prenylation, its transcripts were quantified by qPCR in whole insects, along with those of other genes involved in this pathway. CfGGPPS was expressed throughout insect development and the abundance of its transcripts covaried with that of other prenylation-related genes. Our qPCR results suggest that geranylgeranylation is the predominant form of prenylation in whole C. fumiferana.
Crystal structure of Rab6A'(Q72L) mutant reveals unexpected GDP/Mg <sup>2+</sup> binding with opened GTP-binding domain
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Citation Excerpt :
The Ras-superfamily of small G proteins is a family of GTP hydrolases that includes more than 100 members in eukaryotes from yeast to human [1]. The activity of this family is regulated by the GTP binding state: GDP-bound inactive and GTP-bound active forms [2]. Most of this family of proteins are ubiquitously present inside cells and are key components to various cellular processes, including cytoskeletal organization, mitogenesis, vesicle trafficking, and nuclear transport [3].
The Ras small G protein-superfamily is a family of GTP hydrolases whose activity is regulated by GTP/GDP binding states. Rab6A, a member of the Ras superfamily, is involved in the regulation of vesicle trafficking, which is critical for endocytosis, biosynthesis, secretion, cell differentiation and cell growth. Rab6A exists in two isoforms, termed RabA and Rab6A′. Substitution of Gln72 to Leu72 (Q72L) at Rab6 family blocks GTP hydrolysis activity and this mutation usually causes the Rab6 protein to be constitutively in an active form. Here, we report the crystal structure of the human Rab6A′(Q72L) mutant form at 1.9 Å resolution. Unexpectedly, we found that Rab6A′(Q72L) possesses GDP/Mg²⁺ in the GTP binding pockets, which is formed by a flexible switch I and switch II. Large conformational changes were also detected in the switch I and switch II regions. Our structure revealed that the non-hydrolysable, constitutively active form of Rab6A′ can accommodate GDP/Mg²⁺ in the open conformation.

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Small GTP-Binding Proteins

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Trends Pharmacol. Sci.

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