GPCRs, G Proteins, and Their Impact on β-cell Function

Abstract

Glucose-induced (physiological) insulin secretion from the islet β-cell involves interplay between cationic (i.e., changes in intracellular calcium) and metabolic (i.e., generation of hydrophobic and hydrophilic second messengers) events. A large body of evidence affirms support for novel regulation, by G proteins, of specific intracellular signaling events, including actin cytoskeletal remodeling, transport of insulin-containing granules to the plasma membrane for fusion, and secretion of insulin into the circulation. This article highlights the following aspects of GPCR-G protein biology of the islet. First, it overviews our current understanding of the identity of a wide variety of G protein regulators and their modulatory roles in GPCR-G protein-effector coupling, which is requisite for optimal β-cell function under physiological conditions. Second, it describes evidence in support of novel, noncanonical, GPCR-independent mechanisms of activation of G proteins in the islet. Third, it highlights the evidence indicating that abnormalities in G protein function lead to islet β-cell dysregulation and demise under the duress of metabolic stress and diabetes. Fourth, it summarizes observations of potential beneficial effects of GPCR agonists in preventing/halting metabolic defects in the islet β-cell under various pathological conditions (e.g., metabolic stress and inflammation). Lastly, it identifies knowledge gaps and potential avenues for future research in this evolving field of translational islet biology. Published 2020. Compr Physiol 10:453-490, 2020.

Keywords: cell physiology; diabetes; endocrinology; hormone receptors; intracellular signaling; metabolism.

Figure 1. Schematic representation of GPCR-G protein-effector coupling.

Binding of a ligand to an appropriate GPCR leads to activation of a heterotrimeric G protein via GDP/GTP exchange on the α-subunit. The GTP-bound form of the α-subunit (Gα_.GTP) is functionally active and dissociates from the βγ subunit complex for activation of variety of effector proteins. The GSα_.GTP and G_iα_.GTP promote activation and inactivation of adenylyl cyclases, respectively, leading to either increased or decreased generation of cAMP levels intracellularly. G_qα_.GTP has been implicated in activation of PLases leading to the generation of biologically active hydrophobic (e.g., DAG) and hydrophilic (IP₃) second messengers of insulin secretion. DAG and IP₃ regulate protein kinase C and mobilization of intracellular calcium from the intracellular pools, respectively. Activation of G_12α.GTP results in the regulation of downstream signaling pathways, including activation of Rho subfamily of G proteins, which have been shown to play critical modulatory roles in vesicular transport and actin cytoskeletal remodeling. Lastly, the βγ complex exerts regulatory effects on a wide variety of signaling pathways, including activation of PLases and adenylyl cyclases.

Figure 2. Activation-deactivation of heterotrimeric G proteins.

In its inactive conformation, a heterotrimeric G protein remains as α_.GDP/βγ complex. Following activation by a GPCR (Figure 1), the α_.GTP dissociates from βγ complex to precisely control their respective effector proteins. Exchange of GDP for GTP is mediated via a variety of GEFs. Likewise, the hydrolysis of GTP bound to the Gα subunit is regulated by a number of regulatory proteins and factors (see text for additional details). GEF, GTP/GDP exchange factor; GAP, GTPase-activating protein; R, receptor.

Figure 3. Regulatory roles of small G proteins in glucose-stimulated insulin secretion.

A large body of experimental evidence supports the key roles of small G proteins in the stimulus-secretion coupling of GSIS from the pancreatic β-cell. Shown here are examples of some of these G proteins and their regulatory roles in mediating actin cytoskeletal remodeling, priming/docking, and fusion of insulin-laden secretory granules with the plasma membrane for exocytotic secretion of insulin. Published evidence suggests sequential activation of these G proteins in the cascade of events leading to GSIS. For example, it was demonstrated that GSIS involves successive activation of Arf6 (~1 min), Cdc42 (~3 min), and Rac1 (~15 min) (90). In addition to these, several groups of proteins have been identified and characterized in the islet β-cell, which are essential for recognition and docking of the secretory vesicles on the plasma membrane (127, 298).

Figure 4. Activation-deactivation of small G proteins.

Small G proteins (e.g., Cdc42 and Rac1) undergo activation (GTP-bound) and deactivation (GDP-bound) cycles. Inactive (GDP-bound) G proteins are converted to their active (GTP-bound) counterparts by GEFs. Following transmission of signals requisite for the effector activation, the active form of a candidate G protein returns to its inactive form; such a transition is mediated by the GTPase activity intrinsic to the G protein. In addition, a class of regulatory proteins, referred to as the GTPase-activating proteins (GAPs), has been shown to accelerate the GTPase activity. Lastly, the GDP-dissociation inhibitors (GDIs) play key roles in maintaining the candidate G proteins in their GDP-bound (inactive) configuration. GDIs also contribute toward the targeting of G proteins to appropriate subcellular compartments (see text for additional details). GEFs, GTP/GDP exchange factors; GDI, GDP-dissociation inhibitor; GAP, GTPase-activating protein.

Figure 5. Examples of regulators of small G protein functions in the islet β-cell.

A depiction of various regulatory factors of G protein function which are identified in the pancreatic β-cell. In addition to GEFs, GAPs, and GDIs, several other G protein regulatory proteins/factors (listed under “others”) have been identified and studied in the pancreatic β-cell. Their modulatory roles are described in the narrative.

Figure 6. Proposed model for NDPK-mediated, GPCR-independent (noncanonical) activation of heterotrimeric G proteins.

Based on the available evidence we propose a mechanism for an alternate, GPCR-independent (noncanonical) activation of heterotrimeric G proteins in the pancreatic β-cell by glucose. Trimeric G proteins remain inactive as a α_.GDP/βγ complex. NDPK/histidine kinases catalyze phosphorylation of the β-subunit at a histidine residue (via a phosphoramidate linkage). This phosphate, in turn, is relayed to Gα_.GDP to yield its GTP-bound (active) conformation. This is analogous to the classical ping-pong mechanism of high-energy phosphate transfer mediated by NDPK family of enzymes. Upon activation, Gα_.GTP dissociates from the βγ complex; both of these entities regulate their respective effector proteins, leading to activation or inactivation of downstream signaling pathways (e.g., activation/inactivation of adenylyl cyclase for the generation or suppression of cAMP production). Following this, the GTPase activity, which is intrinsic to the Gα subunit, hydrolyzes the GTP bound to the α-subunit to GDP (inactive conformation), thus facilitating the complexation of the βγ subunit with Gα_.GTP. G, guanosine group.

Figure 7. Examples of noncanonical modulators of heterotrimeric G proteins.

Depicted herein are potential alternate (noncanonical) regulators of heterotrimeric G proteins. Binding of ligands to their cognate GPCRs leads to activation of G proteins via GTP/GDP exchange. Recent studies have identified several non-GPCR-dependent regulatory mechanisms for GTP/GDP exchange including those involving NDPKs. In addition to these, several other modes of noncanonical activation of trimeric G proteins have been described (see text). These include Ric-8A and GEMs (e.g., GIV/Girdin). The GEM class of proteins has been shown to regulate signaling pathways downstream to cell surface receptors. Furthermore, a novel class of regulatory proteins, namely activators of G protein signaling (AGS), has also been identified and studied well in other cell types, but not in the context of islet β-cell function. Altogether, these regulators are involved in GTP/GDP exchange culminating in the formation of Gα_.GTP (active) for effector activation. In a manner akin to the canonically activated trimeric G proteins, the βγ subunits regulate various effectors. Following effector activation, the active form of Gα is converted to its inactive GDP-bound conformation through the intermediacy of intrinsic GTPase. In addition to this, a novel class of regulators of G protein signaling (RGS) has been identified in many cell types, including the islet β-cell. The RGS proteins serve as GAPs in accelerating the GTPase activity, thereby terminating the GPCR activation signals. The identity and functional roles of various RGS proteins are highlighted in the text and in Table 2.

Figure 8. Posttranslational modifications of small G proteins and γ-subunits of trimeric G proteins.

(A) Mevalonic acid is the precursor for the biosynthesis of FPP and GGPP. FTase catalyzes the farnesylation of small G proteins (e.g., Ras), certain γ-subunits of trimeric G proteins, and nuclear lamins A and B. GGTase-I mediates the geranylgeranylation Rho subfamily of G proteins including Rho, Cdc42, Rac1, and Rap1. GGTase-II catalyzes geranylgeranylation Rab subfamily of G proteins. (B) First, FTase or GGTase mediates incorporation of prenyl pyrophosphates into the C-terminal cysteine of G proteins. Second, three amino acids after the farnesylated or geranylgeranylated cysteine are removed by a protease (Rce1). Third, isoprenylcysteine carboxyl methyltransferase (ICMT) catalyzes methylation of the carboxylate group of the prenylated cysteine using S-adenosyl methionine as the methyl donor (131, 137). Not shown in the schematic are additional posttranslational modification steps (e.g., palmitoylation) at a cysteine residue, which is upstream to the prenylated cysteines of G proteins (e.g., Ras and Rac1). This figure is reproduced with permission from Elsevier (137).

Figure 9. Our working model for mechanisms underlying glucose-induced G protein-mediated insulin secretion.

Based on the data accrued in multiple investigations, we propose a model for NDPK/HK signaling step in the stimulus-secretion coupling of GSIS in the islet β-cell. Glucose metabolism leads to activation of PLases (e.g., PLase-C and -D), resulting in the generation of biologically active hydrophobic as well as hydrophilic lipid messengers, which, in turn, activate protein histidine kinases. Furthermore, these biologically active lipids have been shown to regulate GTP-binding and GTPase activities of G proteins in various subcellular fractions derived from the islet. In addition, these second messengers have been shown to promote translocation and membrane association of small G proteins, such as Rac1. Furthermore, activation of protein histidine phosphorylation, by NADPKs, leads to increase in GTP/GDP and GTP channeling to G proteins for their activation. Lastly, findings from our earlier studies suggested novel roles for protein histidine phosphorylation in the regulation of enzymes of glucose metabolism, such as mitochondrial succinyl-coA synthase (121). Based on this experimental evidence, we propose that protein histidine phosphorylation contributes to activation of multiple signaling events in the cascade of events leading to GSIS.

Figure 10. Proposed mechanisms for ROS-dependent regulation of islet β-cell function in normal health and under the duress of metabolic stress.

Several lines of evidence implicate positive and negative modulatory roles for ROS in islet function. NADPH oxidases (Noxs; e.g., Nox1, Nox2, and Nox4) represent the key sources for ROS generation in the islet β-cell. Under physiological conditions, a tonic increase in the intracellular ROS leads to activation of Raf-Ras signaling pathway, which is upstream to ERK1/2 and Rac1 activation, leading to GSIS (149). It has been suggested that intracellularly generated ROS could regulate a variety of cellular events including actin cytoskeletal remodeling (276) and proliferation (171). Therefore, a tonic increase in ROS might promote cytoskeletal remodeling, insulin secretion, and cell proliferation events in the islet β-cell. On the other hand, excessive ROS generation, mediated via activation of various Nox isoforms under the duress of metabolic stress and exposure to pro-inflammatory cytokines, manifests in increased intracellular oxidative stress resulting in the constitutive activation of Rac1 and subsequent activation of downstream stress kinases (JNK1/2, p38, and p53), resulting in cell demise via accelerated apoptosis (131, 250).

Figure 11. Proposed cross talk between various signaling proteins in the GPCR-G protein-effector signalome culminating in physiological insulin secretion.

GPCR agonists and fatty acids bind to their cognate receptors on the plasma membrane, leading to the activation of respective heterotrimeric G proteins (Figure 1). Exposure of the islet β-cell to stimulatory glucose leads to its metabolism and activation of signaling pathways, including activation of NDPKs (and other signaling proteins; Figures 7 and 9), culminating in the activation of trimeric G proteins in a noncanonical mode. Activation of GPCR-G protein signaling pathway results in the generation of second messengers of insulin secretion, including cAMP, IP₃, DAG, and biologically active phospholipids (e.g., lyso-phospholipids). These, in turn, promote activation of small G proteins belonging to Rho and Rab subfamilies, leading to cytoskeletal remodeling, vesicular transport, and fusion of secretory granules with the plasma membrane and secretion of insulin via exocytosis. As discussed in this article, activation of G proteins (heterotrimeric and small G proteins) is controlled precisely by a wide array of regulatory factors, including GEFs, GAPs, GDIs, and others (e.g., AGS proteins, GEMs, and RGS proteins). Furthermore, functional activation of G proteins and their regulators appears to be mediated precisely via posttranslational modifications, including phosphorylation, prenylation, carboxylmethylation, palmitoylation, ubiquitination, and SUMOylation. Not depicted in this figure are potential possibilities for defects in the functional activation of these regulators (e.g., mutations) under the duress of metabolic stress that could result in abnormalities in cross talk within this signalome, resulting in the apoptotic demise of the islet β-cell, and the onset of diabetes.