GPCRs that Rhoar the Guanine nucleotide exchange factors

ABSTRACT

Cell migration, a crucial step in numerous biological processes, is tightly regulated in space and time. Cells employ Rho GTPases, primarily Rho, Rac, and Cdc42, to regulate their motility. Like other small G proteins, Rho GTPases function as biomolecular switches in regulating cell migration by operating between GDP bound ‘OFF’ and GTP bound ‘ON’ states. Guanine nucleotide exchange factors (GEFs) catalyse the shuttling of GTPases from OFF to ON state. G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors that are involved in many signalling phenomena including cell survival and cell migration events. In this review, we summarize signalling mechanisms, involving GPCRs, leading to the activation of RhoGEFs. GPCRs exhibit diverse GEF activation modes that include the interaction of heterotrimeric G protein subunits with different domains of GEFs, phosphorylation, protein–protein interaction, protein–lipid interaction, and/or a combination of these processes.

KEYWORDS:

• Cell migration
• guanine nucleotide
• exchange factors (GEFs)
• Rho GTPases
• G protein-coupled receptors (GPCRs)
• cell signalling

Introduction

Cell migration is a fundamental event in various biological processes such as embryogenesis, immune surveillance, homoeostasis, wound repair, and tissue formation[1]. Cells can migrate as individuals or as a collective group of cells. Collective cell migration contributes to angiogenesis, lumen formation, and tumour cell invasion[2]. On the other hand, individual cell migration is exhibited by cells like fibroblasts (during wound healing) and leukocytes (during immune response)[1]. To initiate migration, cells receive a variety of migratory and invasive cues from their surroundings, which could be in the form of chemicals such as chemokines[3], growth factors (EGF, IGF-1) [4] or sometimes physical signals such as temperature and pressure [5,6]. The migration cycle of an individual cell includes polarization of the front and rear end of the cell, protrusive structure formation at the front end, adherence of the protrusions with the extracellular matrix (ECM), and retraction of the rear end due to contractile force generated by the cytoskeleton[7]. As compared to individual cell migration, collective cell migration is much more complex in terms of its execution and signalling[2]. Similar to individual cell migration, cells moving in a collective fashion also establish front and rear polarity. However, in the collective cell migration, cells at the front end polarize and produce protrusions, whereas, the ones present at the lateral and rear end maintain cell-cell adhesion to generate the required traction force[2]. Thus, the initial steps in all types of cell migration include polarization and formation of protrusions like lamellipodia and filopodia, which require coordinated cytoskeletal dynamics. As cell migration is a vital biological step, it is tightly regulated in space and time by employing a complex signalling network involving the Rho family of small G proteins[8]. These G proteins control cytoskeletal dynamics by regulating actin polymerization. Amongst the known 22 Rho GTPases family members, RhoA, Rac and Cdc42 are the members, most extensively studied for their role in cell migration[8]. RhoA is mostly involved in stress fibre formation and cell contraction [9,10] whereas, Rac controls the formation of lamellipodia, cell spreading, and membrane ruffling [10–13]. Similarly, Cdc42 has been shown to have important roles in filopodia formation and cell reshaping [11,13]. Thus, this Rho GTPase trio lies at the central stage of signal transduction events that control cell migration. Like all canonical G proteins, Rho GTPases exert signalling events by being biomolecular switches. They are active (ON) when bound to GTP and inactive (OFF) when bound to GDP. Two different classes of proteins, Guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) help to shuttle the GTPases between ON and OFF states. GEFs activate GTPases by exchanging their bound GDP with GTP, whereas, GAPs bring them to OFF state by promoting their intrinsic GTP hydrolysis [14] (Figure 1a). For Rho GTPases, there exist another level of regulation in terms of Guanine nucleotide dissociation factors (GDIs), which sequester GDP bound GTPases in the cytosol and replenish them back when required by the cell [15] (Figure 1a). Many reviews excellently summarize the regulation of Rho GTPases [8,14,16–18] by GEFs, GAPs, and GDIs. However, how exactly the migratory cues propagate from various sources to the respective GEFs, GAPs, and GDIs is not well understood. In many instances, the migratory information propagates from the plasma membrane which is communicated largely through membrane-bound proteins such as G protein-coupled receptors (GPCR), Receptor tyrosine kinase (RTK), integrin, and cytokine receptors [16,19,20]. Amongst these, GPCRs are known to be the largest class of membrane receptors that play a key role in the regulation of GEFs. These seven transmembrane receptors are associated with heterotrimeric G proteins, comprising Gαβγ subunits and upon receiving the appropriate extracellular cues, they exert the signalling process by releasing the Gα and Gβγ subunits into the cytosol which in turn regulate their downstream effectors[21]. Aberration in these signalling events is implicated in various pathological conditions such as metabolic disorders, immunological disorders, neurodegenerative diseases, infectious diseases, and cancer [22–24]. A broad variety of signalling cascades initiated by GPCRs during cell migration is discussed earlier by Cotton et al. [25]. However, in this review, we will try to summarize how migratory signals from GPCRs specifically activate RhoGEFs, in the context of cell migration. The mode of propagation of these signals includes, binding of GPCR activated heterotrimeric G protein subunits with different domains of GEFs, phosphorylation of GEFs by various kinases downstream of GPCRs, protein–protein interactions, and protein–lipid interactions.

Figure 1. Schematic representation of some of the GEFs discussed in this review showing intra-domain interaction rendering them in autoinhibitory states. (a) GTPase switching mechanism between ON and OFF state. Canonical GEFs such as (b) p115-RhoGEF and (c) PDZ-RhoGEF are proposed to exhibit autoinhibition due to intra-domain interaction involving their RH domain. Similarly, autoinhibition of (d) AKAP-Lbc and (e) p114-RhoGEF is due to intra-domain interaction involving its non-RH, C terminal region. (f) In p63RhoGEF and (g) Vav GEF, the PH domain is suggested to be responsible for its autoinhibition. (h) In P-Rex GEF, the C terminal domains like IPI4, PDZ1, and PDZ2 are responsible for intra-domain interactions. (i) In TIAM1, the initial 50 amino acid domain (N50), N terminal PH domain (PHn) and the coiled coil extension (CC-Ex) domain of TIAM1 interact with each other to render the GEF to its autoinhibitory state. (j) In non-canonical GEFs like DOCK proteins interaction between N terminal SH3 domain and C terminal domains and/or DHR2 domain is responsible for their autoinhibition.

GPCR mediated heterotrimeric G protein subunit interaction with RH domains of GEFs

Cell polarization, protrusions, and focal adhesions at the site of leading edge formation in both, the individual cell migration as well as collective cell migration (in leader cells) are regulated by RhoA, Rac as well as Cdc42 GTPases [2,7]. Membrane proteins such as chemokine, cytokine, GPCRs, and adhesion receptors activate these GTPases through their GEFs[19]. RhoGEFs can be classified into two categories as canonical and non-canonical (Docker GEFs). Almost all canonical GEFs comprise catalytic Dbl homology domain (DH) and at least one pleckstrin homology (PH) domain[26]. PH domain is primarily involved in the regulation of catalytic activity and also promotes membrane localization of GEF by interacting with membrane lipids like Phosphotidylinositides (PIPs) [27–29]. In a few GEFs, other domains like Ras binding domain (RBD), additional PH domains, regulatory G protein signalling homology (RH) domain, and PDZ domain, are also known to regulate their catalytic activity [30–32]. Most of the canonical GEFs are known to exist in an autoinhibited state due to the interaction between domains at their N and C terminus (Figure 1b-J), making the catalytical domain inaccessible for the GTPase binding. Unlike canonical GEFs, in non-canonical GEFs the PH and DH domains are replaced by DHR-1 and DHR-2 domains (also known as Docker domains), which perform functions similar to their canonical counterparts. Some of the Dedicator of cytokinesis (DOCK) family members are also known to possess additional domains like SH3 or PH domains [33–35]. Similar to the canonical GEFs, DOCK family GEFs are known to exist in an autoinhibitory state[36]. For example, N terminal SH3 domain of DOCK180/DOCK1 is known to interact with its DHR2 domain, to render it inactive[37].

In both types of GEFs, activation happens upon release of their autoinhibitory state either through protein–protein interactions or structural changes on account of post-translation modifications like phosphorylation, which disrupt the intra-domain interactions in them [29,38,39]. However, for many GEFs, the activation mechanism is still not clear. In few instances, GPCRs are known to activate GEFs either by promoting interaction between the heterotrimeric G protein subunits with them or indirectly through pathways regulated by these subunits [40–42]. In some examples, GEFs are known to be activated due to their direct interactions with different domains of GPCRs[43]. In this section, we will discuss ligand-mediated signalling through GPCRs that activate canonical GEFs like leukaemia-associated Rho GEF (LARG), p115-RhoGEF, and PDZ-RhoGEF through heterotrimeric G protein subunit interaction with the regulator of G protein signalling homology (RH) domain of these GEFs.

Leukaemia-associated Rho GEF (LARG) is a canonical GEF, shown to be expressed in most of the cells including peripheral blood leukocytes, spleen, prostate, testis, ovary, small intestine, colon with a minimal expression in thymus [44] and specifically, activates RhoA (Table 1). In mouse embryonic fibroblasts (NIH-3T3), GPCRs, M1 muscarinic cholinergic receptor (M1-mAChR) and Mas related receptor activate RhoA specific GEF LARG, through direct binding of Gα_q subunit (Figure 2a). Similarly, G protein-coupled receptor 132 (G2AR), when triggered with its lipid ligand, activates LARG through Gα₁₃[40]. From co-immunoprecipitation studies, it has been shown that the RH domain of LARG interacts with Gα_q and Gα₁₃ subunits of GPCRs complex to quell its autoinhibitory state[40]. Similarly, in mouse smooth muscle cells, activation of Sphingosine 1 phosphate receptor 2 (S1P₂R) is shown to promote their motility, which is induced by the interaction between LARG and Gα_12/13 subunit [45] (Figure 2b). Analogous LARG activation mechanism is seen in human prostate cancer cells (PC-3) and Human embryo kidney (HEK293T) cells where the Thrombin receptor (PAR) operates through PAR/Gα₁₃/LARG pathway. In mouse embryonic fibroblasts LARG is activated through Lysophosphatidic acid receptor (LPAR)/Gα_12/13/LARG/RhoA pathway involving GPCR, LPAR [46,47] (Figure 2b). LARG is also found to localize with Microtubule organizing centres (MTOC), indicating that the extracellular signals through GPCRs might be involved in maintaining microtubule dynamics and cell polarity through the same Gα_12/13/LARG/RhoA pathway[47].

Table 1. Overview of canonical and non-canonical GEFs discussed in this review, their downstream Rho GTPases, tissue-specific expression and activating GPCRs

Guanine nucleotide exchange factors (GEFs)	GTPase specificity	Tissue Specificity		Activating GPCR	References
		High expression	Moderate/low expression
GPCR mediated heterotrimeric G protein subunit interaction with RH domains of GEFs
LARG	RhoA	Peripheral blood leukocytes, spleen, prostate, testis, ovary, small intestine and colon	Thymus	mAChR, Mas related receptor, G2A, S1P2, PAR and LPA	40,44,45,46,47
PDZ-RhoGEF	RhoA and Cdc42 (through non-RH domain)	Brain, testis, heart, ovary, central nervous system (CNS) and placenta	Kidney, pancreas, spleen, prostate, colon, skeletal muscle, lung and liver	GRPR, LPA1R, LPA2R and CXCR4	46,48,49,52,53,56,53,54
p115-RhoGEF	RhoA	Haematopoietic cells		LPAR, CXCR4, S1PR and β2AR	50,55,56,56
Activation of GEFs involving interaction between heterotrimeric G protein subunits and non-RH domains of GEFs
AKAP-Lbc	RhoA	Cardiac tissues		AT1R and LPAR	54,55,57
p63RhoGEF	RhoA, RhoB, and RhoC	Heart and brain		M3R and H1R	64,65,67
p114-RhoGEF	RhoA and Rac1	Kidney and pancreas	Liver, skeletal muscle and testis	M3R and LPAR	59–61,58
P-Rex1	Rac1/2/3, Cdc42 and RhoG	Neutrophils, macrophages, platelets, endothelial cells, neurons and brain tissues	Bone marrow, thymus, spleen, lymph nodes and lungs	LPA1R, CXCR4 and fMLPR	74,87,59,60,61
Activation of GEFs through phosphorylation, protein-lipid and protein-protein interactions mediated by GPCRs
αPIX	Rac1 and Cdc42	Heart, skeletal muscle and immune cells		fMLPR	13,82,62,63
βPIX	Cdc42 and Rac1	Ubiquitous		LPAR	13,76,82,64
Vav1	Rac1/2, RhoG, and Cdc42	Haematopoietic cells	Pancreas, the tooth enamel, and the trophoblast layer	CXCR4 and fMLPR	42,87,89,65,66
Vav2	RhoA, RhoG, Rac1, and Cdc42	Broadly expressed		CXCR4	42,88,89
Vav3	RhoA, RhoG, and Rac1	Broadly expressed, highest in brain and haematopoietic cells		fMLPR	87,89,67
TIAM1	Rac1	Brain, testis and epidermis		LPA1R and S1P1R	12,99
TIAM2	Rac1	Only restricted to neuronal cells		?	99,100,68
Activation of non-canonical GEFs through GPCR by phosphorylation, protein-lipid and protein-protein interactions
DOCK180/DOCK1	Rac1	Broadly expressed		CXCR4 and LPA	76,69,106,134
DOCK2	Rac1/2	Haematopoietic cells		fMLPR	110,70,71

Figure 2. Schematic representation of signalling pathways which leads to activation of GEFs through interaction between heterotrimeric G protein subunits and RH domains of GEFs. (a) GPCRs, Mas receptor and M1-mAChR activate the GEF, LARG, through Gα_q binding to its RH domain. (b) Activation of LARG through GPCRs, LPAR & S1P₂R occur through direct interaction with Gα_12/13 whereas, GPCRs, G2A & PAR activate LARG through Gα₁₃ subunit. (c) PDZ-RhoGEF mediated RhoA activation is established by GRPR, LPAR, and CXCR4 triggered Gα₁₃ heterotrimeric subunit binding to the RH domain of PDZ-RhoGEF. (d) GPCRs, LPAR, CXCR4, and S1P₂R activate p115-RhoGEF through interaction with Gα₁₃ heterotrimeric subunit. (e) Upon activation of β₂AR, p115-RhoGEF is triggered by β-arrestin activation through Gα_iβγ signalling.

PDZ-RhoGEF and p115-RhoGEF provide examples for Gα₁₃ subunit induced activation of GEF, where the heterotrimeric G subunit interacts with their RH domain to release their autoinhibitory state [38,48]. However, GPCRs that provide the heterotrimeric G subunit (Gα₁₃) for GEF activation differ from cell to cell. C-X-C chemokine receptor type 4 (CXCR4), LPAR and Gastrin-releasing peptide receptor (GRPR) activate RhoA in breast cancer cell lines (MCF7, MDA-MB-231), PC-3 cells and colon cancer cells (Caco-2), respectively, through Gα₁₃/PDZ-RhoGEF/RhoA pathway [38,46,49] (Figure 2c). As seen in PDZ-RhoGEF, p115-RhoGEF also activates RhoA signalling in lymphocytes through Gα₁₃/p115-RhoGEF/RhoA upon CXCR4, LPAR, and S1PR activation [50] (Figure 2d). These are the examples where G protein subunits, Gα_12/13, bind directly to the RH domain containing GEFs to activate them. An exception is seen for p115-RhoGEF in renal cell carcinoma 7 (RCC7) where activated GPCR, β2-adrenergic receptor (β2AR), triggers the GEF indirectly through βArrestin2⁵⁵ (Figure 2e). Furthermore, there are examples where PAR and LPA receptors in PC-3 cells are known to specifically activate LARG and PDZ-RhoGEF, respectively. However, the mechanism underlining their specificity is yet to be determined[46]. Recently it has been shown that prostaglandin receptor activated Gα_s binds to the PH and DH domains as well as their linker region of PDZ-RhoGEF. This interaction of Gα_s with PDZ-RhoGEF renders the GEF active towards Cdc42 [51,52] (Figure 3a).

Figure 3. Schematic representation of signalling pathways which leads to activation of GEFs through interaction between heterotrimeric G protein subunits and non-RH domains of GEFs. (a) Stimulation of prostaglandin receptor activates Gα_s, which in turn binds to PDZ-RhoGEF through DH, PH domains, and their linker region of GEF, thus making it active towards Cdc42. (b) AKAP-Lbc dependent activation of RhoA, by LPAR and AT₁R, happens through the interaction of Gα₁₂ with the C terminus of AKAP-Lbc. (c) and (d) Activation of Gβγ (through M₃R and LPAR) and Gα₁₂ triggers p114-RhoGEF, where Gβγ interacts with the C terminus of DH-PH domain of p114-RhoGEF. (e) p63RhoGEF mediated triggering of RhoA upon activation of M₃R and H₁R receptors. This involves the interaction of Gα_q/11 towards the PH domain of p63RhoGEF. (f) Stimulated GPCRs, LPAR, and CXCR4 activate P-Rex1 through the interaction of the Gβγ subunit with the IP4P domain and the tandem PDZ domains of P-Rex1. This may further lead to the activation of ELMO/DOCK2 through P-Rex activated RhoG. (g) Activation of ET₁R triggers βPIX mediated Cdc42 through the interaction of Gα_q with βPIX.

Activation of GEFs involving interaction between heterotrimeric G protein subunits and non-RH domains of GEFs

G proteins of certain GPCRs are also known to activate GEFs in RH domain independent manner. Examples of these GEFs include A-kinase anchoring protein (AKAP)-Lbc, p114-RhoGEF, p63RhoGEF, Trio, and P-Rex. Details of activation mechanisms of these GEFs by GPCRs will be discussed further. Tissue-specific expression of aforementioned GEFs is given in Table 1. Activation of LPA receptor and Angiotensin 1 receptor (AT₁R) promotes RhoA mediated signalling in HEK293 cells and adult ventricular fibroblasts (AVFs) through Gα₁₂/AKAP-Lbc/RhoA pathway [72,73] (Figure 3b). Here, the interaction of AKAP-Lbc with Gα₁₂ is independent of the RH domain, as this protein lacks this domain[74]. However, the binding of Gα₁₂ to the GEF depends on a 106 amino acid residue region, stretching from residues 2567–2672 towards the C terminus of AKAP-Lbc[41]. Studies in breast cancer cell lines (MDA-MB-435) show that, through a feedback loop signalling, α6β4 integrin regulates LPA induced AKAP-Lbc/Gα₁₂/RhoA pathway to promote activation of Rac GTPase[75]. AKAP-Lbc is predominantly localized in the cytoplasm where it is known to regulate spatial distribution of Protein kinase A (PKA) activity during cell migration [72,76].

The next example involving non-RH domain GEF interacting with heterotrimeric G protein includes p114-RhoGEF, activated by GPCRs, M3-muscarinic receptor (M₃R), and LPA receptor operating in HEK293 and NIH3T3 cells, respectively. In these pathways, the Gβγ subunit of G protein interacts with the DH-PH domain of p114-RhoGEF to bring about its activation, eventually leading to RhoA and Rac signalling [77,78] (Figure 3c). Apart from Gβγ, the Gα₁₂ subunit is also found to interact with p114-RhoGEF (Figure 3d), however, it binds to a region spanning residues from 686 to 791, towards the C terminus of the DH-PH domain [41,77,79]. Furthermore, some evidence suggest that p114-RhoGEF gets triggered both in GPCR dependent and independent manner. For example, it has been shown that tissue-specific apical cell constriction in invertebrates is brought about by non-conserved GPCRs such as Mist (mesoderm-invagination signal transducer) and Smog through the activation of p114-RhoGEF[80]. However, in vertebrates the apical cell constriction process, involving the same p114-RhoGEF, takes place in a GPCR independent manner. In this instance, another GEF DAPLE, which possess Gα-binding-and-activating (GBA) motif could directly activate Gα_iβγ resulting in stimulation of p114-RhoGEF through the Gβγ subunit[81].

Another non-RH domain containing GEF, p63RhoGEF, preferentially expressed in heart and brain tissues, is known to interact with the Gα_q/11 subunit. Here the agonist-stimulated GPCRs like M3-cholinoceptor (M₃R) and the histamine H1 receptor (H₁R), triggers the GEF to activate Gα_q/11/p63RhoGEF/RhoA pathway [82,83] (Figure 3e). Crystal structure of Gα_i/q -p63RhoGEF-RhoA shows an amphipathic helical extension of PH domain (residues 471–485) of the GEF interacting with Gα_i/q [83,84]. Thus, it can be proposed that the autoinhibitory state of the GEF due to the inter-domain interaction invloving PH domain [85,86] is released upon its engagement with Gα_i/q. Furthermore, in fibroblast and COS-7 cells, it is shown that M₃R activated p63RhoGEF competes with phospholipase C (PLCβ) for binding Gα_q[83]. Similar to p63RhoGEF, the Triple function domain protein (Trio) also contains an inhibitory C terminal extension beyond its PH domain[85]. Trio exhibits dual Rac/RhoA GEF activity[87]. Gα_q subunit renders Trio active towards RhoA by binding the inhibitory PH domains and hence relieving its autoinhibition[85]. However, the GPCR which is involved in this pathway has not yet been identified.

In MCF-7 breast cancer cells, chemotactic GPCRs, CXCR4 and LPAR, activate Rac through the Gβγ/PIP3-dependent Rac exchanger1 (P-Rex1)/Rac pathway (Figure 3f). Here the Gα_q or Gα₁₃ but not Gα_s or Gα_i form stable complex with Gβγ. Therefore, the activation of P-Rex1 could be through CXCR4 and LPAR, preferably via Gα_iβγ dependent fashion [88–90]. From cryo-electron microscopy structure it has been suggested that Gβγ interaction with P-Rex1 is a multidomain assembly involving IP4P and tandem PDZ domains of the GEF[91]. Operating under the same pathway, P-Rex1 is also known to activate RhoG leading to the RhoG/ELMO/DOCK2 dependent activation of Rac [92] (Figure 3f).

Activation of GEFs through Phosphorylation, protein–lipid, and protein–protein interactions mediated by GPCRs

Phosphorylation is another means through which GPCRs activate GEFs, wherein these receptors activate downstream kinases that in turn trigger GEFs. One such example is p21-activated kinases interacting exchange proteins (PIX) which, exist in two isoforms αPIX and βPIX and are known to activate Cdc42 and Rac1 GTPases[13]. αPIX is predominantly expressed in haematopoietic and muscle cells whereas, βPIX is expressed ubiquitously. Splice variants of these proteins are majorly found in the brain [13,93]. It has been shown that activation of βPIX involves Gα_i of LPA receptor in HeyA8 ovarian cancer cell lines, where Gα_i recruits Src kinase, which phosphorylates Tyr-442 residue of βPIX to activate the GEF [94–96] (Figure 4a). Apart from Src, other kinases like p21-activated kinases (PAK) are also known to phosphorylate numerous serine and tyrosine residues of βPIX, especially the residues at its PH, GITI-binding, N-terminal spacer region (amino acid 61–99), and C terminal coiled-coil domains. Other than GEF activation, phosphorylation at many of these sites could have important roles in regulating βPIX interaction with other signal transduction proteins[97]. Another kinase, PKA, is shown to phosphorylate βPIX primarily at Ser-516 and Thr-526, when triggered through Endothelin 1 receptor (ET₁R) induced Gα_s subunit[98]. Furthermore, βPIX is also known to interact with Endothelin 1 receptor (ET₁R) activated Gα_q subunits to trigger Cdc42⁹⁹ (Figure 3g). Though these studies suggest that the direct binding of the Gα_q subunit is required for βPIX activation[99]. But, the role of phosphorylation in this case is not clear.

Figure 4. Activation of GEFs through Phosphorylation, protein-protein, and lipid-mediated interactions involving downstream of GPCR signalling. (a) Upon LPAR stimulation, activation of βPIX takes place through, Gα_i mediated Src kinase activation, which further phosphorylates βPIX. (b) Activation of fMLPR triggers Cdc42 through Gβγ-PAK1-αPIX and Rac1 through Gβγ-PAK1-αPIX-GIT2 sequential complex formation, where activation of αPIX takes place through PAK1 binding. Cdc42 and Rac1 are involved in feedforward mechanism where αPIX and Rac1 enhance PAK1 activity. Here activation of Cdc42 enhances GEF activity of αPIX towards Rac1. (c) CXCR4 activates Vav1 and Vav2 through JAK signalling. (d) Activation of Rac through fMLPR stimulation involves concerted signalling between Vav1/3 and P-Rex GEFs. (e) LPAR triggers Rac1 through PKC and CamKII mediated phosphorylation of TIAM1. (f) Stimulation of GPCRs, LPAR and S1P₁R, activate TIAM through Gα_iβγ/PI3K/TIAM1/Rac1 pathway, where TIAM1 interacts with a PI3K lipid product, PIP3. (g) Protein–protein interaction mediated triggering of RhoA when activated Frizzled7 recruits Dishevelled (Dvl) and Daam-1 proteins which activate the Rho WGEF.

Although relatively little is known about phosphorylation induced activation of αPIX by PAK, it has been suggested that PAK triggers this GEF by releasing it from its inactive dimer state [13,100]. This pathway involves activation of Gβγ through formyl-methionyl-leucyl-phenylalanine receptor (fMLPR), which further triggers a cascade of signalling events, propagating through p21-activated kinase 1 (PAK1). Further, PAK1 interacts with the SH3 domain of the GEF to release αPIX from its dimeric state to its active monomeric state which further triggers Cdc42 [13,100]. Activated Cdc42 is also known to enhance the affinity of this GEF towards Rac1 (Figure 4b). Thus, it appears that there is a mutual feedforward mechanism between these two GTPases. On one hand, binding of Cdc42 to one of the DH domains of the dimeric αPIX introduces conformational changes in the GEF to enhance its activity towards Rac1¹⁰⁰[101], whereas, on the other hand, in a feedforward mechanism, αPIX enhances kinase activity of PAK1 to further activate Cdc42[102]. Furthermore, αPIX is also known to trigger Rac1 in fMLPR controlled pathway. Here, fMLPR triggers to form a quaternary complex Gβγ-PAK1-αPIX-GIT2 in a sequential manner to recast its specificity for Rac1 (Figure 4b). During this pathway, the role of phosphorylation in the activation of αPIX is not clear, but it has been shown that activation of αPIX could take place through binding to phosphatidylinositol 3, 4, 5-trisphosphate (PIP3), a lipid product of PI3-kinase[103]. Similar to Cdc42, Rac1 is also suggested to operate in the feedforward cycle to further enhance the activity of PAK1 towards the GEF[104].

Vav family of GEFs provide another example for activation of GEFs, through phosphorylation relay triggered by GPCRs like CXCR4 and fMLPR [42,105]. This family of GEF consists of 3 members, namely, Vav1 (specific to Rac1/2, RhoG, and Cdc42), Vav2 (specific to Rac1, RhoA, RhoG, and Cdc42), and Vav3 (specific to Rac1, RhoG, and RhoA) [106,107] and their tissue-specific expression is shown in Table 1. Out of these GEFs, Vav1 and Vav2 are activated by pathways involving Janus kinase2 (JAK2), Janus kinase3 (JAK3) and the receptor involved in this particular signalling is CXCR4[42]. It is interesting to know that activation of this GEF through JAK is independent of GPCR-Gα_i pathway [42,108] (Figure 4c). In P1V1 neutrophils, through an unknown mechanism, fMLPR leads to activation of P-Rex1 and Vav1/Vav3¹⁰⁵ GEFs (Figure 4d). However, the role of GPCRs, CXCR4 and fMLPR, in the activation of Vav proteins is not clear. For Vav1 it has been surmised that fMLPR could activate Src or Syk kinases through downstream signalling to trigger this RacGEF [109,110].

The T-cell lymphoma invasion and metastasis inducing factor 1 (TIAM1) provides a good example of phosphorylation mediated activation of GEF. Through Small-angle X-ray scattering (SAXS) based model it was suggested that the initial 50 amino acid domain (N50), N terminal PH domain (PHn), and the coiled-coil extension (CC-Ex) domain of TIAM1 interact with each other to render the GEF inactive[32]. Release of this autoinhibition involves multiple steps including phosphorylation of TIAM1 by protein kinase C (PKCα) and Ca2+/calmodulin-dependent protein kinase II (CamKII) (Figure 4e). In Swiss 3T3 fibroblast cells, this phosphorylation pathway is triggered by the LPA receptor[111]. Based on various experimental studies it has been proposed that sequential phosphorylation of Ser29 and Ser33 of TIAM1 by atypical C kinase (aPKCγ)[32], followed by phosphorylation of Tyr829 by TrkB kinase[112], disrupt the intra-domain interactions of the GEF to make it active [32,113]. Apart from phosphorylation, there exist other modes of TIAM1 activation including protein–protein interactions involving PHn-CC-Ex domain of TIAM1 with Par3 in the Par polarity complex (Par3, Par6 and aPKC) [32,114,115].

It has also been proposed that lipid-mediated interactions too can activate TIAM1. For example GPCRs, LPA₁R and S1P₁R, induced Gα_iβγ/PI3K/TIAM1/Rac1 pathway promote binding of PI3K lipid product, phosphatidylinositol 3, 4, 5-trisphosphate (PIP3)[116], to the PH domain of the GEF to make it active [12,117] (Figure 4f). However, complete activation of TIAM1 requires additional signals from Gα_iβγ[12]. Although TIAM2/STEF is relatively less characterized compared to TIAM1, it has been suggested that the activation of this GEF is also a complex multistep process. Forster resonance energy transfer based biosensor studies on dibutyryl cAMP (dbcAMP) treated PC12D cells has revealed that the phosphorylation of Thr-749 by dbcAMP activated PKA at the plasma membrane enhances the GEF activity of TIAM2 towards Rac1[118].

Activation of GEFs through direct interaction of GPCR domains

Few reports suggest that GPCRs could activate GEFs by directly interacting with them, instead of utilizing the heterotrimeric G proteins. For example, PDZ-RhoGEF and LARG interact with the C terminus of LPA₁R and LPA₂R receptors through their PDZ domains (Figure 5a) to get activated[43]. Similarly, activation of Vav2 involves interaction of the C terminus of the Parathyroid hormone receptor (PTHR) with its DH and PH domains. It is interesting to note that Vav2 competes with PTHR to bind Gα_q subunit and also interferes with the PTHR signalling[119] (Figure 5b). Other than these examples, there is not much information on how GPCRs could directly activate GEFs.

Figure 5. Schematic representation of activation of GEFs upon direct interaction with GPCR domains. (a) GEFs, LARG, and PDZ-RhoGEF activate RhoA signalling upon interacting with the C terminus of LPAR through their PDZ domains. (b) Similarly, Vav2 interacts with the C terminus of PTHR through its DH and PH domains.

Non-canonical GPCRs activate GEFs through the scaffold protein Dishevelled (Dvl)

There are about 11 non-canonical GPCRs identified as Frizzled (FZD) class receptors. Other than smoothened (SMO), all other members of this family are activated by secreted glycoproteins, Wingless/Int1 (Wnt). There are 19 Wnts identified in humans. A combination of interactions between FZDs and Wnts is known to trigger diverse signalling pathways classified as canonical and non-canonical Wnt signalling pathways[120]. However, the exact mapping between specific Wnt-FZD interactions to a particular pathway is not clear. The common feature in the non-canonical FZD GPCR pathway is the recruitment of a scaffold protein dishevelled (Dvl) at the cytosolic end of the receptor upon binding of Wnts at their extracellular domain. It has been shown that Dvl forms higher oligomers to act as a signalling node, known as signalosome to further propagate the signal[121]. With reference to GEF activation, Frizzled 7 (FZD7) is known to activate RhoA through WGEF (Weakly similar to Rho GEF 5). In Xenopus embryos, it has been shown that activation of FZD7 leads to recruitment of Dvl-2, which subsequently interacts with Dishevelled-associated activator of morphogenesis 1 (Daam-1) protein. This signal further propagates to activate WGEF. However, the exact mechanism of activation is not clear¹²² (Figure 4g). It is interesting to note that WGEF is found to interact directly with the PDZ domain of Dvl-2 and with the N terminal domain of Daam-1 leading to its activation. Thus, it has been suggested that FZD7 could operate through multiple modes to activate WGEF[122].

Activation of non-canonical GEFs through GPCRs

As mentioned previously, the non-canonical Dedicator of cytokinesis (DOCK) family GEFs, also known as Docker GEFs, contain DHR-1 and DHR-2 domains, in place of PH and DH domains of canonical GEFs. Similar to canonical GEFs, Docker GEFs are known to be activated through phosphorylation, protein–protein interactions, and/or lipid-mediated interactions. In HL60 and HeLa cell lines, activation of the chemokine receptor, CXCR4, triggers activation of Rac through Gβγ/ELMO1/DOCK180/Rac1 pathway. Similarly, in MDA-MB-231 cell lines, CXCR4 activates Rac through Gα_i/ELMO1/DOCK180/Rac1 pathway⁶⁹[123], (Figure 6a). In both of these pathways Gβγ and Gα_i subunits were found to interact with ELMO1, wherein Gα_i interacts with N terminus of ELMO1,⁶⁹[123], leading to conformational changes in the ELMO-DOCK complex, resulting in the release of DOCK from its autoinhibitory state. Another mechanism for DOCK180 activation by LPA is through a sequential complexation of Gα_i2-src-p130Cas/ELMO/DOCK180⁹⁴ [124,125] (Figure 6b), in which Src kinase is shown to phosphorylate DOCK180 at Tyr1811, enhancing the interaction of DOCK180 with CrkII and p130Cas proteins, which activates Rac[126].

Figure 6. Schematic representation of activation of non-canonical GEFs by GPCRs. (a) CXCR4 activates ELMO1/DOCK180 through the interaction of ELMO1 with Gβγ and Gα_i subunits. (b) Stimulation of LPAR leads to Rac activation through Gα_i2-Src-p130Cas/ELMO/DOCK180/Rac1 pathway during which Src is proposed to phosphorylate DOCK180 at Tyr1811. (c) ELMO/DOCK2 mediated Rac activation takes place through binding of DHR-1 domain of DOCK2 to lipid, PIP3 upon fMLPR activation. (d) ELMO1 and ELMO2 are shown to interact with the C terminal of the BAI1 receptor through their N-terminal fragments to trigger the DOCK/Rac pathway.

DOCK2, another member of the DOCK family is shown to be activated through lipid-mediated interaction. This process involves fMLPR induced association of DHR-1 domain of DOCK2 with phosphatidylinositol 3, 4, 5-trisphosphate (PIP₃) in a PI3K dependent manner [127,128], wherein interaction with PIP₃ triggers GEF activity of DOCK2 towards Rac (Figure 6c). It has been also suggested that activation of chemokine receptors triggers DOCK2 through Gα_i subunit, but the exact mechanism is unclear[129]. In apoptotic cells, Brain-specific angiogenesis inhibitor 1 receptor (BAI1) is shown to trigger ELMO/DOCK/Rac signalling (Figure 6d). These signalling events depend on the interaction of cytosolic fragment of BAI1 (aa 1431–1582) with N terminal fragments of ELMO1 (aa 1–558) and ELMO2 (aa 1–520), which subsequently binds to DOCK1/DOCK2 [130,131]. Although the activation of non-canonical GEFs is similar to canonical GEFs, the pathways that operate are markedly different.

Summary

Activation of GTPases like RhoA, Cdc42, and Rac is a crucial step in cell migration, as they are the key players controlling the cytoskeletal rearrangement. This process is highly regulated in space and time by employing GEFs and GAPs. GEFs provide one of the important steps of regulation, wherein they switch the GTPases ‘ON’ by exchanging GDP with GTP. Based on sequence and structure GEFs can be classified as canonical and non-canonical. The common feature of both canonical and non-canonical GEFs is that they exist in an autoinhibitory inactive state (Figure 1b-J) and release of this autoinhibitory state is essential for them to activate their cognate GTPases. The GEF activation process is done through multiple modes like phosphorylation of GEFs or interaction of GEFs with a different scaffold or activating proteins or through lipid interactions. In this review, we have consolidated how GPCRs play a crucial role in activating GEFs by exerting these activation mechanism. These events include the interaction of GEFs (through their RH and non-RH domains) with heterotrimeric G protein subunits activated by GPCRs, activation of kinases that phosphorylate GEFs, and protein/lipid interactions that release GEFs from their autoinhibited state. Complete understanding of these signalling events at the atomic level is crucial to decipher signalling processes, as this would be highly rewarding in addressing pathological conditions like cancer metastasis, immunological disorders, and neurodegenerative diseases.

Although the field of cell signalling is rapidly evolving, there are significant gaps in our understanding of signalling events that control cell migration, especially with reference to the role of GPCRs. Majority of the efforts, to date, are on mapping the key players involved in the signal transduction, which is largely driven by gene knockout/silencing and protein pull-down studies. However, the key questions like how in real-time the signals transverse from GPCRs to the downstream proteins? how concomitantly operating diverse pathways synchronize to exert cell migration? and how few GPCRs autonomously operate to control divergent pathways? remain unaddressed. Perhaps, innovative real-time imaging techniques, involving bioluminescence resonance energy transfer and fluorescence resonance energy transfer would help to dissect the system. Although some efforts have been made in this direction [132–135], given the emerging complexity of the system, unravelling the signalling events that control cell migration remains challenging.

Acknowledgments

KK thank the ICGEB Research Grant [CRP/IND15] for the grant and AO would like to acknowledge the Council of Scientific & Industrial Research, India for fellowship.

Disclosure statement

The authors declare no conflicts of interest.

Additional information

Funding

This work was supported by the International Center for Genetic Engineering and Biotechnology [CRP/IND15].