Multiple Guanine Nucleotide Exchange Factors Mediate Glucose-Induced Rac1 Activation and Insulin Secretion: Is It Precise Regulatory Control or a Case of Two Peas from the Same Pod?

Abstract

Glucose-stimulated insulin secretion involves G protein (Rac1)-mediated cytoskeletal remodeling and vesicular transport and fusion with the plasma membrane. Recent evidence implicates at least three guanine nucleotide exchange factors (GEFs), namely, Tiam1, Vav2, and P-Rex1, in glucose-induced activation of Rac1 and insulin secretion. This Viewpoint highlights potential mechanisms underlying Tiam1/Vav2/P-Rex1 sensitive Rac1-mediated insulin secretion in the glucose-stimulated β-cell.

Glucose-induced insulin secretion (GSIS) from the pancreatic β-cell involves a variety of metabolic and cationic events, including generation of adenine and guanine nucleotides, hydrolytic products of phospholipids, and changes in intracellular calcium levels. Burgeoning evidence implicates novel regulatory roles for G proteins (trimeric and monomeric) as mediators of GSIS.^1,2 In the context of small G proteins, earlier studies have demonstrated critical regulatory roles of members of the subfamilies of ADP-ribosylation factors (Arf6), Rho (Cdc42, Rac1), and Rab (Rab3, Rab27) in the cascade of events leading to GSIS.^1,2 It is noteworthy that activation–deactivation cycles of these G proteins involve precise control by a variety of regulatory factors, including the guanine nucleotide exchange factors (GEFs), the GTPase-activating proteins (GAPs), and the guanine nucleotide dissociation inhibitors (GDIs). Recent reviews have described potential identity and regulatory roles of these factors in islet β-cell function, including GSIS.^1,2 From a functional standpoint, GEFs mediate activation of G proteins by promoting the release of GDP to enable GTP binding. GAPs catalyze the hydrolysis of GTP-bound to G proteins culminating in their inactivation. GDIs are involved in the sequestration of G proteins thereby preventing their activation by GEFs. Interestingly, a large number of GEFs (82 members), GAPs (69 members), and GDIs (3 members) are expressed in mammalian cells highlighting the complexity of G-protein-mediated regulation of cells in health and disease. Indeed, several recent studies have investigated regulatory roles of multiple GEFs, GDIs, GAPs in β-cell function in health and in metabolic stress conditions.¹

Ras-related C3 botulinum toxin substrate 1 (Rac1), a small G protein (focus of the current Viewpoint), has been shown to regulate a variety of β-cell functions, including cytoskeletal remodeling and the transport of insulin-laden secretory granules to the plasma membrane for fusion and secretion of insulin via exocytosis. Using a variety of molecular biological (active and inactive mutants, siRNA) and pharmacological (small molecule inhibitors) approaches, several studies have reported requisite roles for Rac1 in GSIS (reviewed in refs (1,2)). These findings were further confirmed in islets derived from animal models in which Rac1 is deleted conditionally in pancreatic β-cells.³ As depicted in Figure 1, Rac1 undergoes activation–deactivation steps mediated by GEFs, GAP, and RhoGDIα. Briefly, in a glucose-stimulated β-cell, the GDP-bound (inactive) Rac1 (retained in this conformation by RhoGDIα) is converted to its GTP-bound conformation by at least three GEFs (Tiam1, Vav2, P-Rex1), leading to its activation and optimal regulation of its effector proteins (NADPH oxidase, PAK, etc.) and associated cellular events (actin cytoskeletal remodeling) resulting in exocytotic secretion of insulin. Following completion of these events, the GTP-bound Rac1 is converted to its GDP-bound configuration via the intermediacy of a GAP (MgRac1GAP) and its regulatory adaptor protein (APPL2) followed by its reassociation with RhoGDIα.⁴ The following is a brief summary on roles of three GEFs (Tiam1, Vav2, and P-Rex1) in the signaling events leading to GSIS.

Figure 1.

Signaling steps involved in glucose-mediated activation of Rac1 and insulin secretion. Glucose metabolism leads to GEF-mediated activation of Rac1 (Rac1-GTP), which, in turn, promotes activation of signaling steps necessary for GSIS. Active Rac1 is converted to its inactive conformation through the intermediacy of MgRac1GAP/APPL2 facilitating in complexation of GDP-bound Rac1 to RhoGDIα.

Original studies from our laboratory have investigated regulatory roles for T-cell lymphoma invasion and metastasis-inducing protein 1 (Tiam1), a specific nucleotide exchange factor (GEF) for Rac1, in GSIS in pancreatic β-cells.⁵ We reported that NSC23766, a specific inhibitor of Tiam1-mediated activation of Rac1, markedly inhibited glucose-induced Rac1 activation and insulin secretion in INS-1 832/13 cells and rat islets. siRNA-mediated depletion of Tiam1 significantly suppressed glucose-induced membrane trafficking and activation of Rac1 in INS-1 832/13 cells. It is noteworthy that, in contrast to the inhibitory effects of NSC23766, siRNA-mediated knockdown of Tiam1 potentiated GSIS in INS-1 832/13 cells; such an effect on GSIS was sensitive to extracellular calcium. On the basis of this evidence, we proposed a modulatory role for Tiam1-Rac1 axis in GSIS. We also pointed out a potential Tiam1-Rac1-independent but calcium-sensitive mechanism for GSIS in these cells.⁵

In follow-up studies, we demonstrated key roles of Vav guanine nucleotide exchange factor 2 (Vav2), in glucose-induced Rac1 activation, actin remodeling, and insulin secretion in pancreatic β-cells.⁶ Ehop-016, a small molecule inhibitor of the Vav2-Rac1 signaling module, or siRNA-Vav2 markedly suppressed glucose-induced Rac1 activation and GSIS in INS-1 832/13 cells. Inhibitory effects of Ehop-016 were confirmed in primary rat islets. Microscopic evidence suggested increased colocalization of Vav2 with Rac1 under conditions of GSIS. Real-time imaging in live cells demonstrated a significant attenuation of glucose-induced cortical actin remodeling by Ehop-016. On the basis of these findings, we concluded that Vav2 is involved in glucose-induced Rac1 activation, actin remodeling, and GSIS in pancreatic β-cells.⁶

Lastly, more recent studies from our laboratory have explored the roles of PIP3-dependent Rac1 exchange factor (P-Rex1) signaling pathway in the stimulus secretion coupling of GSIS.⁷ We observed that siRNA-mediated knockdown of P-Rex1 attenuated glucose-induced Rac1 activation, membrane association, and insulin secretion. LY294002, a known inhibitor of PI3-kinase, potentiated GSIS without affecting glucose-induced Rac1 activation. On the basis of these findings, we surmised that P-Rex1 plays a novel regulatory role in glucose-induced Rac1 activation and insulin secretion.

Findings from the above investigations raise an important question of why does the islet β-cell need more than one GEF to activate Rac1 and insulin secretion under physiological conditions? Potential possibilities for such a multifaceted regulatory control of Rac1 in the cascade of events leading to GSIS are discussed below.

Data from studies addressing roles of Tiam1-Rac1⁵ and P-Rex1-Rac1⁷ signaling modules in GSIS have suggested that both of these GEFs play significant roles in membrane trafficking of Rac1 under conditions favorable for GSIS to occur. These findings highlight novel properties of these GEFs, which may be independent of their GEF functions. It is widely felt that dissociation of G protein-GDI complex following cellular activation drives the GDP-GTP exchange mediated by GEFs. Along these lines, it has been demonstrated in insulin-secreting cells that specific second messengers (e.g., biologically active lipids) generated during glucose metabolism promote dissociation of Rac1 from its complex with RhoGDIα, resulting in its association with the membrane for optimal interaction with effector proteins leading to insulin secretion.¹ These events might include, but are not limited to, association of Rac1 with membranous core of phagocyte-like NADPH oxidase (Nox2), which is a requisite for the formation of Nox2 holoenzyme that mediates tonic increase in reactive oxygen species levels, to promote actin cytoskeletal remodeling and GSIS.¹ Therefore, based on the observations that both P-Rex1 and Tiam1 facilitate membrane association of Rac1, it may be necessary to undertake additional investigations to further affirm their roles in membrane trafficking of G proteins, such as Rac1.

It is noteworthy that recent proteomics-based studies by Marei and co-workers have identified distinct sets of interacting partners for Rac1 following its activation by Tiam1 and P-Rex1.⁸ More importantly, investigations from these researchers have demonstrated requisite roles for P-Rex1 and Tiam1 in promoting Rac1-mediated anti- vs pro-migratory effects. Based on compelling data accrued from multiple experimental approaches, these researchers have proposed novel temporal and spatial regulatory roles of these GEFs in eliciting their effects on Rac1 and its associated downstream signaling events.⁸ Likewise, multi-GEF mediated activation of small G proteins (Rac1/2, RhoG) have been described in neutrophils.⁹ Future proteomics investigations may be necessary for the identification of Rac1 interactome in pancreatic β-cells under conditions of its activation by each of these GEFs (Tiam1, Vav2, and P-Rex-1) leading to GSIS. Such approaches are warranted specifically in the light of the contributory roles of Tiam1 and Vav2 in the sustained activation of Rac1 under conditions of metabolic stress.¹⁰ Furthermore, data derived from studies, involving live cell imaging approaches, have highlighted critical roles for Vav2-Rac1 axis in glucose-induced filamentous actin remodeling in β-cells.⁶ It may be necessary to further assess the roles of signaling events (and Rac1-effector protein complexes) derived from Tiam1-Rac1 and P-Rex1-Rac1 modules in cytoskeletal rearrangements and vesicle fusion leading to insulin release.

Recent investigations have uncovered novel noncanonical regulation of P-Rex1, which is mediated via binding of the regulatory subunit of protein kinase A (i.e., PKA-Riα) to P-Rex1 leading to activation of its GEF function. This has been proposed, but not tested in the context of P-Rex1-Rac1 mediated GSIS.¹¹ Furthermore, it may be likely that activation of specific trimeric G proteins via noncanonical mechanism(s) might promote P-Rex1-Rac1 axis leading to GSIS. As proposed recently,¹ such mechanisms might include nonreceptor-mediated functional activation of individual subunits of trimeric G proteins, such as post-translational carboxyl methylation of specific G_γ-subunits and/or histidine phosphorylation of the G_β-subunits leading to the activation of the putative trimeric G proteins that couples downstream signaling proteins, such as Phosphatidyl inositol-3-kinase (PI3-kinase) for the activation of P-Rex-1-Rac1 signaling pathway culminating in GSIS. Future studies will test this experimental model.

In conclusion, GEF-mediated regulation of small G protein (Rac1) and associated downstream signaling events is highly complex. As reviewed recently by Marei and Malliri, at least 20 GEFs have been shown to regulate Rac1 functions.¹² Evidence highlighted herein suggests that at least 3 GEFs (Tiam1, Vav2, and P-rex1; Figure 1) control Rac1 functions in pancreatic β-cells. I propose that GSIS might underlie a well-orchestrated cross talk between G proteins (trimeric and monomeric) and their regulatory factors at multiple subcellular compartments (cytoplasm, plasma membrane, mitochondria, nucleus, etc.) within the pancreatic β-cell. Additional studies, including in vivo studies involving islets derived from mouse models in which the GEFs are conditionally deleted in β-cells, as well as investigations aimed at identification of specific interacting partners of each of these GEFs (Tiam1, Vav2, P-Rex-1, and potentially others) with specific G proteins (e.g., Rac1) that could contribute to the generation of specific signals to trigger cytoskeletal remodeling and secretion of insulin. Lastly, given the known roles of some of these GEFs (Tiam1 and Vav2) in the sustained activation and inappropriate targeting of Rac1 to nucleus (mislocalization) leading to dysregulated β-cell under metabolic stress,¹ it may be necessary to further identify the interacting partners and related signaling pathways for these GEFs under these experimental conditions, specifically in β-cell models of impaired insulin secretion and diabetes.

Glossary

Abbreviations

APPL2

Adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 2

ARF6

ADP-ribosylation factor 6

GAP

GTPase-activating protein

GDI

Guanine nucleotide dissociation inhibitor

GEF

Guanine nucleotide exchange factor

GSIS

Glucose-stimulated insulin secretion

GTP

Guanosine triphosphate

Nox2

Phagocyte-like NADPH oxidase

PI-3-kinase

Phosphatidyl inositol-3-kinase

P-REX1

PIP3-dependent Rac1 exchange factor

PKA

Protein kinase A

Rac1

Ras-related C3 botulinum toxin substrate 1

ROS

Reactive oxygen species

Tiam1

T-cell lymphoma invasion and metastasis-inducing protein 1

Vav2

Vav guanine nucleotide exchange factor 2

This research is supported by a Merit Review Award (BX004663) from the U.S. Department of Veterans Affairs and the National Institutes of Health (EY022230). A.K. is the recipient of a Senior Research Career Scientist Award (K6 BX005383) from the U.S. Department of Veterans Affairs.

The author declares no competing financial interest.