Ubiquitous Gα_s is an activator of adenylate cyclase that produces messenger cAMP from ATP. In its quiescent state, Gα_s is associated with Gβ/γ dimer. The intrinsic GTPase activity of Gs causes its deactivation and reassociation with Gβ/γ dimer. In its inactive form, Gs binds to Gβ1/γ1, Gβ1/γ2, Gβ1/γ5, Gβ1/γ7, Gβ2/γ5, and Gβ2/γ7 [781].

Subunit Gα_s is activated by G-protein-coupled receptors, such as β-adrenergic, glucagon, luteinizing hormone, calcitonin, and dopamine D₁ and D₅ receptors [781]. Subunit Gs^GTP activates adenylate cyclase isoforms. Affinity of Gs for both Gβ/γ and adenylate cyclase is increased by N-terminal palmitoylation.

Several types of Gα_i exist (Gα_i1–Gα_i3) that mainly inhibit the cAMP-dependent pathway, but activate the phospholipase-C pathway. In fact, the Gi/o subclass is composed not only of Gα_i, but also Gα_o, Gα_gust, and Gα_t (transducin) proteins (Table 9.3).

Cells respond to growth factors by either migrating or proliferating. Cell movement and proliferation are indeed mutually exclusive. Growth factor receptors, such as EGFR, VEGFR, and PDGFR receptors, can actually trigger cell motion or division according to the type and concentration of the signaling ligand on the one hand and the density and distribution of receptors on the other. During cell migration, phospholipase-Cγ1 and phosphatidylinositol 3-kinase and its effector protein kinase-B are stimulated and coupled to actin remodeling at the leading edge. In proliferating cells, another set of signals (ERK1 and ERK2, Src kinase, and STAT5b) is involved for the activation of nuclear transcription factors and DNA synthesis. Girdin (girders of actin filaments)⁹ is an actin-binding PKB substrate¹⁰ that operates in actin organization and PKB-dependent cell motility, at least, in fibroblasts. It is phosphorylated upon stimulation by insulin-like growth factor IGF1 that fosters tumor cell movement [782].¹¹ Girdin is a guanine nucleotide-exchange factor for G proteins involved in the regulation of the PI3K–PKB axis, actin cytoskeleton remodeling, and cell migration.¹² Proteic subunit Gα_i and its ubiquitous guanine nucleotide-exchange factor girdin orchestrate the migration–proliferation dichotomy downstream from EGFR signaling [784]. Girdin directly interacts with EGFR and associates Gα_i to the receptor. A Gα_i–girdin–EGFR complex indeed assembles, EGFR autophosphorylates (a process necessary for cell migration, but not for mitosis), and EGFR residence in the plasma membrane is prolonged (signaling from the cell surface rather than from endosomes). Plasma membrane-based motogenic signals (PLCγ1 and the PI3K–PKB axis) are triggered and cell migration starts. Activation of Gα_i by girdin is required in cell migration primed by EGF and insulin during epithelial wound healing, macrophage chemotaxis, and tumor cell migration. In addition, cancer angiogenesis, invasion, and metastasis also rely on girdin mediator following VEGF and IGF stimulation. On the other hand, a GEF-deficient girdin splice variant promotes mitogenic signals and cell proliferation occurs.¹³

Subunit Gα_q links certain types of activated G-protein-coupled receptors to intracellular signaling cascades. Various types of GPCRs couple to Gα_q, such as acetylcholine, angiotensin, catecholamine, endothelin, glutamate, histamine, lysophospholipid, and serotonin receptors [789] (Tables 9.5 to 9.8).

The subclass of ubiquitous Gα_q subunits include 4 families: Gq, G11, G14, and G16 (G15 and G16 are murine and human orthologs, respectively; Table 9.9).

Members of the subclass 4 of Gα subunits, i.e., G12 and G13, participate in various cellular functions. They activate Rho GTPases. Both Gα₁₂ and Gα₁₃ trigger similar cellular effects that lead to gene transcription, reorganization of the actin cytoskeleton, formation of actin stress fibers, and assembly of focal adhesions, events that are associated with RhoA activation [800]. Nonetheless, Gα₁₂ and Gα₁₃ do not have only redundant activities. Subunit Gα₁₃, but not Gα₁₂, contributes to the development of the vasculature. Factors RhoGEF1 and RhoGEF12 are more effective for G13 than G12. Whereas Gα₁₃ is able to stimulate RhoGEF1 and unphosphorylated RhoGEF12 in vitro, Gα₁₂ only activates phosphorylated RhoGEF12 [800]. Whereas Gα₁₂ activates RhoA for stress-fiber formation upon activation by G-protein-coupled receptors bound to endothelin, thrombin, and vasopressin, Gα₁₃ primes RhoA signaling as a mediator of GPCRs ligated by bradykinin, serotonin, and lisophosphatidic acid. Consequently, Gα₁₂ and Gα₁₃ can be targeted by distinct receptors. They not only possess different affectors, but also different effectors. Furthermore, they can have opposite effect on a given mediator. Subunit G13 activates the transcription factor NRF2, whereas G12 antagonizes it [800]. Besides, G13 is involved in the regulation of NOS2 expression.

Subunits G12 and G13 not only target small GTPase Rho via RhoGEF, but also phospholipase-Cε and -D, MAPK, and Na⁺–H⁺ exchanger, as G12 and G13 interact with several proteins (Table 9.10). Ezrin, radixin, and moesin connect various receptors, ion channels, and integrins to the cytoskeleton. Four Rho GTPase guanine nucleotide-exchange factors (Sect. 9.4.1.9) are regulated by G12/13 (Table 9.11): (1) RhoGEF1,²² which is produced in blood cells; (2) RhoGEF11,²³ which is widespread, but at low level, except in the central nervous system; (3) ubiquitous RhoGEF12;²⁴ and (4) widespread RhoGEF13.²⁵

In humans, the RGS superfamily contains at least 37 identified proteins. It can be decomposed into many families [811]: (1) A or RZ family with members (RGS17, RGS19, and RGS20) that are characterized by an N-terminus with many cysteine residues, which can be reversibly palmitoylated; (2) B or R4 family (RGS1–RGS5, RGS8, RGS13, RGS16, RGS18, and RGS21); (3) C or R7 family, members (RGS6, RGS7, RGS9, and RGS11) of which can couple Gα subunits to GPCRs in the absence of Gβγ dimers. They indeed bind Gβ5 via Gγ-like (GGl) domain to form heterodimers;²⁷ (4) D or R12 family (RGS10, RGS12, RGS14); (5) E or RA family that is composed of RGS box-containing axin1 and axin2, negative regulators of the Wnt pathway, that interact with adenomatous polyposis coli protein; (6) F or GEF family that comprises RhoA-specific guanine nucleotide exchange factors (RhoGEF1, RhoGEF11, and RhoGEF12); (7) G or GRK family that encompasses RGS box-containing G-protein-coupled receptor kinases (GRK1–GRK7; Sect. 5.2.11); and (8) H or Snx family that contains RGS box-containing sorting nexins (Snx13, Snx14, Snx25). Other RGS groups include [811]: (1) dual-specificity A-kinase anchor proteins AKAP10a and AKAP10b that bind PKA regulatory subunits and (2) RGS22 isoforms (RGS22a–RGS22c).

Most RGS proteins are GPCR inhibitors, either by their GAP activity for Gα or by effector antagonism, as they can bind activated Gα^GTP in competition with effectors. On the other hand, members of the F class of RGS proteins are positive regulators; RhoGEF1, RhoGEF11, and RhoGEF12 (Sect. 9.4.1.9) couple Gα_q, Gα₁₂, and/or Gα₁₃ subunits to monomeric RhoA GTPase (Sect. 9.3.13.1). Owing to their RGS box, they convert inactive RhoA^GDP into active RhoA^GTP. These 3 RGS box-containing, RhoA-specific guanine nucleotide-exchange factors (RGS-RhoGEFs) serve as Gα effectors that couple not only GPCRs, but also semaphorin receptors to RhoA GTPases [811].²⁸ The RGS box of RhoGEF1 serves as a GTPase-activating protein motif Gα₁₂ and Gα₁₃ subunits. Protein RhoGEF12 is a Gα-responsive RhoGEF for Gq, G12 and G13 [811]. However, RhoGEF12 stimulation by Gα₁₂ ^GTP depends on RhoGEF12 phosphorylation by TEC family kinases or focal adhesion kinase. In addition, these 3 RGS-RhoGEFs couple distinct receptors to RhoA activation (RGS-RhoGEF signaling specificity).²⁹

The RGS proteins with a Gα_i∕o–Loco interaction (GoLoco or G-protein regulatory [GPR]) motif, such as RGS12 and RGS14, have a guanine nucleotide-dissociation inhibitor activity, as they slow spontaneous exchange of GDP for GTP and inhibit association with Gβγ subunits (sequestration) [811]. GoLoco motif-containing proteins generally bind to Gα^GDP subunits of the Gi/o subclass. Other GoLoco motif-containing proteins include Rap1GAP (Sect. 9.4.2.3) that accelerates GTP hydrolysis by Rap1 and Rap2-interacting protein (Sect. 9.3.9), as well as G-protein signaling modulators GPSM1,³⁰ GPSM2,³¹ GPSM3,³² which is able to simultaneously bind more than one Gα_i1 subunit, and GPSM4.³³ The GPSM proteins do not modify kinetics or magnitude of effector activation, but can reduce agonist binding affinity, hence signaling intensity [811].

In summary, regulators of G-protein signaling serve as: (1) guanosine triphosphate-accelerating protein for Gα proteins (GαGAP), hence as signaling inhibitors; (2) Gα effectors, such as RGS-RhoGEFs that couple receptors to monomeric GTPase RhoA; (3) signaling scaffolds between signaling mediators, such as RGS12 that operates as a nexus between Gα, small guanosine triphosphatase Ras, and protein kinases; (4) coupling factors between Gα and GPCRs, such as dimers composed of C-class RGSs and Gβ5; and (5) guanine nucleotide-dissociation inhibitors (e.g., RGS12 and RGS14).

Activity of RGS proteins is regulated within a cell. Phosphatidylinositol (1,4,5)-trisphosphate inhibits RGS proteins [812]. Ca–calmodulin restores the GAP activity. Both PIP₃ and Ca–calmodulin bind to the same RGS site. Inhibition and disinhibition of GAP activity of RGS4 by these molecules explain oscillations in intracellular calcium concentration.³⁴ Furthermore, RGS proteins are targeted by different protein kinases.³⁵

The RGS proteins are implicated in interactions involving various signaling molecules associated with G-protein signaling pathways and ion carriers at the plasma membrane, such as Ca, phospholipids (especially phosphoinositides), and Tyr kinases.³⁶ Besides, lipopolysaccharides and angiotensin-2 increase the expression of RGS proteins in vascular cells.

Cardiac RGSs hinder phospholipase-C activity via Gq/11, especially phospholipase-C stimulation of endothelin-1.⁴⁰ Hence, RGS4 has an antihypertrophic effect. Furthermore, cardiac RGSs regulates the activation and deactivation kinetics of Gβγ-gated K⁺ channels, thus the inward rectifier K⁺ channel regulated by acetylcholine via Gi/o (RGSs accelerate GTP hydrolysis rate of Gα_i∕o and regulate ACh-dependent relaxation).⁴¹

Membrane-attached RGS3, a Gβγ-binding protein [815] that is strongly expressed in the heart [816], attenuates signaling not only via Gα_i and Gα_q ∕ 11, but also via Gβγ-mediated signaling (via phospholipase-C, mitogen-activated protein kinase, and phosphatidylinositol 3-kinase). Agent RGS6 is relatively abundant in atriomyocytes [817]. Sorting nexin SNx13,⁴² a Gα_s-specific GAP, inhibits adenylate cyclase stimulation induced by the α-adrenoceptor–Gα_s complex. It may then modulate the activity of cardiac Ca channels. It binds to membrane phosphatidylinositol 3-phosphate, thus participating in early endosome structure. The RhoGEF proteins have Dbl- (DH) and pleckstrin (PH) homology domains. The DH domain is responsible for exchange activity and the PH domain is likely involved in subcellular localization. The RGS-like RhoGEFs act on Gα₁₂ and Gα₁₃ [815]. Coupling of GPCRs to G12/13 in cardiomyocytes is associated with contractility [818], and protein kinase-C–mediated activation of sarcolemmal Na⁺–H⁺ exchangers [819]. GPCR kinases phosphorylate activated GPCR receptors. Agent GRK2, highly expressed in the human heart (as well as GRK5 and GRK6), interacts with Gq/11 (GRK2 sequesters activated Gα_q).

Activator of G-protein signaling AGS1 selectively activates Gi/o independently of G-protein-coupled receptors. Protein AGS1 is a member of the RAS hyperfamily of small GTPases that plays the role of guanine nucleotide-exchange factor for Gi subunit. Protein AGS1 can antagonize GPCR by reducing the pool of G-proteins available for GPCR coupling.

In addition, AGS1 impedes increase in activity of Gβγ-regulated inwardly rectifying K⁺ channel (GIRK) activated by muscarinic M₂ receptors [824]. In neurons, AGS1 interacts with adaptors, such as NOS-associated CaPON and NCK2, as well as nitric oxide synthase NOS1. Adaptor CaPON competes with adaptor DLg4 involved in the ^NMDAGlu–NOS1 pathway to form a ^NMDAGlu–CaPON–NOS1–AGS1 complex [825]. Moreover, AGS1 is expressed in the suprachiasmatic nuclei according to circadian rhythm [826].

Activator of G-protein signaling AGS2 is similar to a light chain component of cytoskeletal and ciliary dynein. AGS2 interacts with numerous signaling effectors, especially GPCRs. Dynein could organize signaling complexes to regulate organelle displacement. AGS8 is produced by ventricular cardiomyocytes (but not in cardiac fibroblasts, aortic smooth muscle cells, and endothelial cells) in response to hypoxia (but not by tachycardia, hypertrophy, or failure) [827]. AGS8 interacts directly with Gβγ without disturbing the regulation of PLCβ2 by Gβγ.

Activator of G-protein signaling AGS3, which is widely expressed (with tissue-specific splicing),⁴⁵ acts on plasmalemmal concentrations of certain receptors and channels by modulating cellular transport of receptors and channels such asK_IR2.1. Moreover, AGS3 activates G protein (Gi inhibition and Gβγ stimulation) in a receptor-independent fashion [828]. AGS3 interacts only with members of the Gα_i∕o subclass (except Gz), i.e., with both Gi and Go, but it is a guanine dissociation inhibitor for Gi3 only (not for Go) [829]. Agent AGS3 binds and stabilizes Gα_i ^GDP, and then blocks reassociation of Gα_i with Gβγ dimer. Therefore, AGS3 inhibits Gα_i, but favors Gβγ signaling.

The superfamily of ARF GTPases includes several families constitued by: (1) ARF isoforms (ARF1–ARF6);⁶⁵ (2) secretion-associated and Ras-related protein SAR1; (3) ARF-related protein ArfRP1;⁶⁶ (4) ARF-like GTPases (ARL1–ARL22);⁶⁷ and (5) adpribosylation factor domain-containing protein-1 (ARD1 or ARFD1) that corresponds to tripartite motif-containing protein TriM23,⁶⁸ a ubiquitin-protein ligase and a GTP-binding protein.⁶⁹

Small ARF GTPases can be classified into 3 subfamilies: subfamily 1 with ARF1 to ARF3 that regulate the assembly of coat complexes onto budding vesicles and activate enzymes that target lipids; subfamily 2 with ARF4 and ARF5 that may operate in Golgi transport and recruitment of coat components to trans-Golgi membranes; and subfamily 3 with ARF6 that controls endosomal-membrane trafficking. Isoform ARF2 is absent in humans [839].

Adpribosylation factors are characterized by their subcellular compartmentation and binding partners that dictates the function of each member.⁷⁰ However, the majority of ARF effectors can interact with several ARF GTPases. All ARF GTPases are myristoylated (second N-terminus Gly), thus allowing ARF tethering to membranes. Activation–deactivation cycle of ARFs, hence association–dissociation of transport vesicle coat proteins, are regulated by ArfGEFs (Sect. 9.4.1.1) and ArfGAPs (Sect. 9.4.2.1).

The primary role of ARF GTPases is the regulation of vesicular membrane transfer, especially the formation of coated vesicles. The ARF proteins actually regulate structure, budding from donor membrane, motion, and fusion with acceptor membrane of vesicles devoted to intracellular transport of cargos, as they recruit coat proteins, prime proteic complex assembly, modulate phospholipid metabolism, contribute to actin remodeling, especially at the cell cortex, and participate in some signaling pathways. Therefore, ARF GTPases are involved in endo- and exocytosis, phagocytosis, cytokinesis, and cell adhesion and migration. Small ARF GTPases not only regulate vesicular motions, but also organelle structure.

Small ARF GTPases execute their function by anchoring to membrane surfaces, where they interact with other proteins to initiate budding and maturation of transport vesicles. They are mainly involved in the vesicular transport of lipids and proteins between the endoplasmic reticulum, where cargos are synthesized, and the Golgi body, where cargos undergo post-translational modification (Table 9.21 and 9.22).

Small ARF GTPases contribute to morphology and location of the Golgi body that depends on the cell cytoskeleton as well as actin cytoskeleton-dependent changes in cell morphology. Golgi-associated ARF1 is involved in the formation of focal adhesions via the delivery of adaptor paxillin to the plasma membrane and Rho activation. Small ARF6 GTPase facilitates recruitment of active Rac to the plasma membrane. Small Rac GTPase alone can promote the migration of epithelial cell sheets during wound healing, but not cell scattering that requires cooperation of ARF6 [841].

The ARF proteins possess a 2-domain structure [842]. The myristoylated N-terminal helix used for membrane binding⁷¹ is separated from the C-terminus by a flexible linker. This linker may yield a certain degree of adaptability in binding modes for the huge quantity of ARF-interacting proteins, in addition to specific binding sites for lipids on some of these small GTPases.

Exchange of GDP by GTP occurs at the ARF myristoyl binding site. Active ARFs promote and stabilize curved surfaces of budding vesicles [842]. C-terminus positioned on the membrane surface provides an interaction structure for activators, adaptors, effectors, and inhibitory GAP proteins. At least certain ARF types (e.g., ARF4 to ARF6) remain bound to membranes in their GDP-bound conformation.

To function, ARFs must interact sequentially or simultaneously with: (1) guanine nucleotide-exchange factors and lipids that catalyze their activation; (2) proteic adaptors that modulate recruitment of cargo into nascent buds; (3) lipid-modifying enzymes; and (4) GTPase-activating proteins, the 2 latter categories being required for budding and maturation of transport carriers.

Partners of ARFs can be grouped into 3 categories: (1) ArfGEFs that have a Sec7 domain (Sect. 9.4.1.1);⁷² (2) ArfGAPs that possess a cysteine-rich ArfGAP domain (Sect. 9.4.2.1);⁷³ and (3) effectors and ARF-binding lipids, either non-specific partners that promote nucleotide-exchange, or specific partners, such as PI(4,5)P₂ and phosphatidic acid [839]. Several GEFs and GAPs interact with ARF proteins. Proteins of the ArfGEF and ArfGAP sets, like ARF, can translocate to membranes in a regulated fashion. Nucleotide-sensitive partners comprise mitotic kinesin-like protein MKlP1, arfaptin-1 and -2, and arfophilins.⁷⁴ In addition to arfophilins, JNK-interacting proteins JIP3 and JIP4 (MAPK8IP3 and MAPK8IP4) that also selectively bind ARF6 mediate postendocytic recycling during cytokinesis. All ARF GTPases share several effectors, such as phospholipase-D and several phosphoinositide kinases (PI4P5Kα–PI4P5Kγ).

Soluble ARFs (ARF1–ARF5) are regulators of vesicular transport, particularly to and from the Golgi body, and endosomes, as well as activators of phospholipase-D. Active ^GTPARF1 to ^GTPARF5 translocate onto membranes where they recruit: (1) ARF-dependent coat proteins AP1, AP3, AP4, GGA13, and CoP1; (2) scaffolding and trafficking Munc18-interactors MInt1 (a.k.a. amyloid-β A4 precursor-binding protein APBa1 and neuron-specific adaptor X11α), MInt2 (a.k.a. APBa2, X11β, and X11-like protein [X11l]), and MInt3 (a.k.a. APBa3, X11γ, and X11-like-2 protein X11l2);⁷⁵ and (3) lipid enzymes, such as phospholipase-D, phosphatidylinositol 4-kinase-3, and phosphatidylinositol 4-phosphate 5-kinase [844].

Adpribosylation factor-like proteins are members of the ARF superfamily. The ARL family include 24 members (Table 9.23).

Generally, ARL proteins bind to cell membranes using an N-terminal helix that is inserted into the lipid bilayer once activated. In addition, this N-terminal helix contains a myristoyl or an acetyl group.

Adpribosylation factor-related protein ArfRP1⁸⁵ is another Ras-related, membrane-associated, monomeric GTPase with some degree of similarity with ARF molecules. Protein ArfRP1 has an unusual feature for an ARF superfamily member: it has a rather high intrinsic GTPase activity. It is mainly associated with the trans-Golgi network [852]. This regulator works synergistically with ARL1 as well as golgin-97 and -245 on Golgi membranes.

In addition, ArfRP1 associates with the plasma membrane. Protein ArfRP1 connects to ArfGEF cytohesin-1 and -2 (Sect. 9.4.1.1. Protein ArfRP1^GTP stimulates phospholipase-D to produce phosphatidic acid, a messenger for vesicle formation and trafficking [853]. Phospholipase-D1a and -D1b reside in the perinuclear region, whereas PLD2 is attached to the plasma membrane and is less responsive to ARFs than PLD1 enzyme. Phospholipase-D2 can be stimulated from activated receptor Tyr kinase and G-protein-coupled receptors that prime ARF-, Rho-, and/or PKC-dependent signaling.

Adpribosylation factor domain-containing protein-1 (ARD1) that pertains to the ARF superfamily of small GTPases corresponds to ubiquitin-protein ligase TriM23 of the tripartite motif (TRIM) family. Members of the TRIM family are implicated in gene transcription, signal transduction, vesicular transport, antiviral defense,⁸⁶ phospholipase-D activation, and protein degradation via ubiquitination. Three alternatively spliced transcript variants have been detected in humans (ARD1α–ARD1γ).

The TRIM motif includes 3 zinc-binding domains, a RING, B-box type 1 and type 2, and a coiled-coil region. Its C-terminus contains an adpribosylation factor (ARF or P3) domain and a guanine nucleotide-binding site. Its N-terminus possesses a GTPase-activating protein (GAP or P5) domain. Protein ARD1 cycles between active (GTP-bound) and inactive (GDP-bound) forms. Unlike ARFs, ARD1 lacks the myristoylation site in its ARF domain. The GAP activity of ARD1 is restricted to its ARF domain, without GAP activity for any other ARFs [854].

Protein ARD1 localizes to lysosomes and the Golgi body. It operates in the formation of intracellular transport vesicles and their displacement between cellular compartments, as well as phospholipase-D activation [854].

Cytohesin-1 (or ARNO2) serves not only as ArfGEF, but also supports GTP binding to ARD1, at least in vitro [854]. Free Mg at concentration in the micromolar range favors guanine nucleotide release from ARD1 protein. In addition, ARD1 interacts with TriM29⁸⁷ and TriM31 Ub ligases.⁸⁸

Small GTPase secretion-associated and Ras-related protein SAR1, a member of the RAS superfamily, operates as a molecular switch to control protein–protein and protein–lipid interactions during vesicle budding from the endoplasmic reticulum. Unlike all Ras GTPases that use either myristoyl or prenyl groups to direct membrane association and function, SAR1 lacks such modifications, but contains a SAR1-NH2-terminal activation recruitment (STAR) motif [855]. The STAR motif mediates the recruitment of SAR1 to endoplasmic reticulum membranes and facilitates its interaction with membrane-associated guanine nucleotide exchange factor Sec12. In addition, an N-terminal motif assists interaction with the GTPase-activating protein complex Sec23/24. Small GTPase SAR1 coordinates CoP2 coat assembly and disassembly and cargo selection with the recruitment of CoP2 coat to initiate export from the endoplasmic reticulum.

Golgins are long coiled-coil proteins that localize to particular Golgi subdomains via their C-termini. GRIP domain-containing golgins (golgin-97 and -245 and GRIP and coiled-coil domain-containing proteins GCC1 or GCC88 and GCC2 or GCC185) bind via their C-termini Arf-like protein ArL1 on the trans-Golgi network as well as Rab2, Rab6, Rab19, and Rab30. These molecules of the trans-Golgi network as well as other compartments of the Golgi body such as cis-Golgi network capture Rab-containing vesicles and exclude ribosomes (sise ∼ 25 nm) [873]. Actin and spectrin form a mesh around the Golgi body that reorganizes for vesicle arrival and departure.

Skeletal muscle is a major site of dietary glucose storage, using insulin-mediated translocation of GluT4 at the plasma membrane via the PKB–TBC1D4 pathway. Agent TBC1 domain-containing protein TBC1D4,¹¹² a RabGAP does not target the same Rab GTPase in skeletal myocytes and adipocytes; Rab8a is involved in GluT4 exocytosis in myocytes and Rab10 in adipocytes. Insulin promotes activation of both Rab8a and Rab13 of the group-8 Rab GTPases, but not Rab10, in rat myocytes, Rab8a activation preceding that of Rab13 [884]. The latter is characterized by a wider intracellular distribution than that of Rab8a GTPase, restricted to the perinuclear region.

Most cells have members of the hyperfamily of small GTPases that are structurally classified into, at least, 5 superfamilies (ARF, Rab, Ran, Ras, and Rho), although expression levels of these members vary. Yet, a few members have a tissue-specific expression. Small GTPase Rab17 is detected only in epithelial cells.

Phosphatidylinositol (4,5)-bisphosphate is produced at sites of N-cadherin-mediated intercellular adhesion due to activated Rac1 GTPase. The latter mediates insertion of transient receptor potential channel TRPC5 into the plasma membrane via its stimulation of PI(4)P5K enzyme.

Isoform Rac1 elicits migration of multiple cell types (e.g., macrophages, T lymphocytes, Schwann cells, epithelial cells, and fibroblasts). Response generated by Rac1 relies particularly on chemotactic fMLP peptide. Both Rac1 and Rac2 regulate cell spreading. Both Rac1 and Rac2 intervene in the actin mesh and spectrin scaffold of erythrocytes. Proteins Rac1, Rac3, and RhoG promote axon growth and guidance.

Sumoylation of Rac1 promotes its activity, hence cell migration. Protein inhibitor of activated STAT PIAS3, a small ubiquitin like modifier (SUMo) ligase, interacts with Rac1, particularly Rac1^GTP, and primes its sumoylation [891].

Isoform Rac2 is required for migration and phagocytosis of neutrophils. Protein Rac2 interacts with phagocyte NADPH oxidase NOx2 [892]. Inactive Rac2^GDP is bound to RhoGDI that sequesters Rac2 and prevents Rac2 from interacting with membranes as well as GEFs, GAPs, and effectors.

Ubiquitous Rac3 has an association rate of GDP similar to Rac1, but 13-fold higher than that of Rac2. The intrinsic GTP hydrolysis rate of Rac3 is similar to those of Rac1 or Rac2, but is about 5-fold faster than that of Ras [893]. GTPase-activating protein Bcr can stimulate GTP hydrolysis of Rac3 to a similar degree to that of Rac1. Like other Rac GTPases, guanine nucleotide-exchange factors such as Tiam1 increases the dissociation rate of GDP on Rac3. Protein Rac3 also interacts with guanine nucleotide-dissociation inhibitors.

Its specific effectors include calcium–integrin-binding protein (CIB), nuclear DNA-binding protein C1D, and nuclear receptor-binding protein (NRBP). Constitutively active Rac3 localizes to internal membranes of the endoplasmatic reticulum and Golgi body. Active ^GTPRac3 can also be detected at the inner leaflet of the plasma membrane and membrane protrusions.

Members of the Rap1 subfamily are able to bind Ras effectors, with an affinity similar to or sometimes higher than Ras, such as cRaf, PI3K, and RalGEFs. They promote activation of certain component of the mitogen-activated protein kinase module, such as bRaf kinase. In addition to their effect on MAPK signaling, Rap1 GTPases participate in cytoskeletal rearrangement and cadherin- and integrin-mediated cell adhesions, as well as the regulation of cell proliferation and differentiation. Both Rap1a and Rap1b contribute to vesicular delivery of E-cadherins with Ral and exocyst complex components ExoC2 and ExoC8, hence controlling plasmalemmal E-cadherin density, as well as cytoskeletal linkage to E-cadherins with catenin-δ1 and Rap1 effector, afadin,¹²¹ and thus cell adhesion stabilization. Afadin also binds to Rap1GAP signal-induced proliferation-associated protein SPA1 (or SIPA1). It can then control integrin-mediated cell adhesion via the recruitment of SPA1 and Rap1^GTP.

Small GTPase Rap1 is also able to block cell transformation owing to its capacity to form non-productive complexes with Ras effectors. Anti-oncogenic effect of Rap1 indeed relies on sequestration of RasA1 (or p120RasGAP) and cRaf kinase. Conversely, Rap1 can cause Rac activation and mediate relocalization of RacGEFs Vav2 and TIAM1 during intercellular adhesion maturation and cell spreading [903].

In hematopoietic cells, Rap1 provokes lymphocyte aggregation, T-cell anergy, and platelet activation. In the nervous system, Rap1 contributes to the regulation of axonal differentiation, dendritic development, dendritic and spine morphology, and synapse remodeling, in particular synaptic depression by removing AMPARs from synapses via P38MAPK [904]. In addition, Rap1 couples cAMP signaling to ERK1 and ERK2 kinases.

Several Rap guanine nucleotide-exchange factors (RapGEF; Sect. 9.4.1.6)) activate Rap GTPases. They include RapGEF1 (or C3G), RapGEF2 (PDZ-GEF), RapGEF3 and -4 (EPAC1 and -2), Rap(Ras)GRPs, and dedicator of cytokinesis DOCK4.

Ras-related GTPases Rap1 and Rap2 are able to interact with RalGEFs, such as Ral GDP-dissociation stimulator (RalGDS or RalGEF), RalGDS-like (RGL GEF), and RalGDS-like factor (RLF) that, all, are effectors of Ras [905]. In the central nervous system, Rap2 specifically interacts with Rap2-interacting protein RpIP8 (or Rundc3a) [906].

Activation of Rap1 follows its interaction with Rap1-specific RapGEF1 protein. Under basal conditions, RapGEF1 is associated with CRK2 adaptor. Stretch of the extracellular matrix is transduced into intracellular biochemical signals. Small Rap1 GTPase provokes integrin-mediated adhesion and then influences actin dynamics. Protein Rap1 activates integrins via Rap1^GTP-interacting adaptor (RIAM) [907].¹²² Cell stretch activates Rap1 using RapGEF1 and CRK2 (Rap1–CRK2–RapGEF1 pathway) [908]. Stretch-induced phosphorylation of CRK-associated substrate (CAS or BCAR1) of the cytoskeleton is due to SRC family kinases. Adaptor CRK2 binds directly to phosphorylated CAS, especially in cell–matrix adhesion sites.

Small Rap1 GTPase is a member of the shelterin complex, or telosome complex, that regulates the length of telomeres and protects chromosome ends [909].¹²³ Shelterin protects chromosome ends from the activity of DNA-repair molecules.¹²⁴ Repressor and activator Rap1 protein¹²⁵ is not involved in the maintenance of telomere length, the organization of telomeric chromatin, and inhibition of non-homologous end joining (NHEJ), a repair process of double-strand DNA breaks at telomeres, but is required for the inhibition of homology-directed repair (HDR) [910].¹²⁶

Small Rap1 GTPase not only protects telomeres from sister chromatid exchange, but also intervenes in transcriptional regulation. Protein Rap1 may bind to telomeric and extratelomeric (TTAGGG)₂ DNA consensus motifs.¹²⁷ It operates in transcriptional regulation and silences genes in proximal and subtelomeric regions [911].

Small Rap1 GTPase also controls the NFκB pathway that mediates the transcriptional response to various types of cellular and developmental signals and stresses [912]. A significant proportion of cytoplasmic Rap1 is constitutively bound to inhibitor of NFκB kinase (IκB kinases [IKK]). Protein Rap1 enables efficient recruitment of IKKs and phosphorylation of P65_NFκB.¹²⁸ Moreover, NFκB may, in turn, regulate Rap1 production via the NFκB-binding site in the RAP1 promoter region.

Proteins Rap2a and Rap2b have a low intrinsic GTPase activity. Like Rap1, Rap2 GTPases contribute to synapse remodeling, particularly removal of AMPA receptors via Jun N-terminal kinases. Besides, neither Rap2a nor Rap2b antagonize Ras GTPases. The Rap2 proteins can also be involved in cell signaling, as they can mediate activation of phospholipase-Cε.

Small Ras GTPases include Ras, Rap, Ral, and others [919]. Small GTPases Rho, Rac, and CDC42 are the 3 best known families of the RHO superfamily. Kinases of the RoCK family are effectors of Rho GTPases. Small Rac and CDC42 GTPases act via P21-activated kinases. Small Rho GTPases regulate cytoskeletal activity during cell motility, shape change, and contraction.¹³¹ Each RHO family is characterized by specific effects on the actin cytoskeleton. Agent Rho is involved in the formation of stress fibers, Rac of membrane ruffles and lamellipodia, and CDC42 of filopodia (radial unipolar bundles). Members of all RHO families also regulate the assembly of integrin-containing adhesion complexes, and thus cell–matrix interactions and cell adhesion.

Members of the Ras family constitute 2 subfamilies. A first subfamily includes Harvey hRas, Kirsten kRas,¹³² and neuroblastoma nRas, whereas related rRas isoforms are grouped in a second subfamily. In mammals, 3 RAS genes generate subfamily-1 Ras GTPases (hRAS, kRAS, and nRAS). The KRAS transcript can be alternatively spliced, thereby producing kRas4a and kRas4b subtypes.

Post-translational modifications are aimed at enabling proper protein folding and localization, signal transmission, as well as protein degradation. Post-translational modifications of Ras GTPases direct them to various cellular membranes and, in some cases, modulate GTP–GDP exchange [920]. These modifications are either constitutive, such as irreversible farnesylation of the C-terminal CAAX motif,¹³³ reversible palmitoylation,¹³⁴ methylation, and proteolysis of a C-terminal propeptide,¹³⁵ or conditional, such as reversible phosphorylation,¹³⁶ peptidyl-prolyl isomerisation,¹³⁷ mono- and diubiquitination,¹³⁸ nitrosylation,¹³⁹ adpribosylation, and glucosylation.

Membrane tethering and trafficking of small GTPases of the P21RAS subfamily other than kRas4b are regulated by reversible palmitoylation of Cys residues in their C-terminus [920].¹⁴⁰ The covalent attachment of the acyl chain of a fatty acid to a protein is called protein acylation. Unlike farnesylation, palmitoylation is reversible. Reversibility is ensured by one or more thioesterases, such as acyl-protein thioesterase APT1. Both hRas and nRas undergo palmitoylation–depalmitoylation (acylation–deacylation) cycles for Ras transfer from (anterograde) and to (retrograde transport) the Golgi body. Palmitoylation is required for the transfer of hRas and nRas from endomembranes to the plasma membrane. Farnesylated Ras has only a slight affinity for membranes, but farnesylated and palmitoylated Ras has a much higher affinity [920]. Mono- and dipalmitoylation control Ras localization.

Cis–trans isomerization of a peptidyl-prolyl bond adjacent to a palmitate in hRAS acts as a molecular timer that regulates depalmitoylation and retrograde transfer.

Phosphorylation of kRas4b in its polybasic region allows kRAS4b dissociation from the plasma membrane. S-nitrosylation of Ras promotes guanine nucleotide exchange.

Mono- and diubiquitination of hRAS regulate its association with endosomes; kRas4b monoubiquitination enhances its activation. Ubiquitination regulates hRas transfer to and from endosomes. Effector of Ras GTPase Ras and Rab interactor RIn1 is required for RabEx5-dependent Ras ubiquitination.

Small Ras GTPases are involved in the regulation of cell growth, differentiation, and survival. They are encoded by genes that experience mutations in about 30% of cancers in humans.

The components of the P21RAS subfamily have a similar effector domain, which interacts with downstream pathway targets. Both Rap and Ras¹⁴¹ can bind the same effectors to regulate intracellular signaling events.

In the GTP-bound conformation, Ras binds to and activates its effectors, members of the RAF family, phosphatidylinositol 3-kinase, and members of the RalGEF family [922] (Table 9.29). Effector of Ras for small Ras-like GTPases RalA and RalB are the guanine nucleotide-exchange factors Ral guanine nucleotide-dissociation stimulator (RalGDS or RalGEF) and RalGDS-like protein. Other effectors include phospholipase-Cε,¹⁴² T-cell lymphoma invasion and metastasis GEF TIAM1,¹⁴³ and Ras interaction/interference protein RIn1, afadin, and Ras association domain-containing family proteins (RASSF) [923].¹⁴⁴