The heterotrimeric G protein G(i3) regulates hepatic autophagy downstream of the insulin receptor

Compelling evidence suggests that the heterotrimeric G protein G(i3) specifically transmits the antiautophagic effects of insulin and amino acids in the liver. This points to a previously unrecognized cross talk between the insulin receptor tyrosine kinase and G(i3). Interestingly, G(i3) is localized not only to plasma membranes but also to membranes of the autophagosomal compartment. Furthermore, as part of insulin's or phenylalanine's actions to inhibit autophagy, G(i3) is redistributed away from autophagosomes. Therefore, endomembrane-associated rather than plasma membrane-localized G(i3) may serve as the target of insulin's endocrine and metabolic actions. We therefore propose that the function and regulation of organelle-associated heterotrimeric G proteins may be different from their roles at the plasma membrane where they act as signal transducers of seven-transmembrane receptors. Here, we discuss recent findings and propose a function for G(i3) in mTOR-dependent signaling pathways. We hypothesize that G(i) family members may have tissue-specific roles in the regulation of autophagy under different physiological and pathological conditions.     

WNT-5A stimulates the GDP/GTP exchange at pertussis toxin-sensitive heterotrimeric G proteins

The lipoglycoproteins of the WNT family act on seven transmembrane-spanning Class Frizzled receptors. Here, we show that WNT-5A evokes a proliferative response in a mouse microglia-like cell line (N13), which is sensitive to pertussis toxin, thus implicating the involvement of heterotrimeric G proteins of the G_i/o family. We continue to show that WNT-5A stimulation of N13 membranes and permeabilized cells evokes the exchange of GDP for GTP of pertussis toxin-sensitive G proteins employing [γ-³⁵S]GTP assay and activity state-specific antibodies to GTP-bound G_i proteins. Our functional analysis of the PTX-sensitivity of WNT-induced G protein activation and PCR analysis of G protein and FZD expression patterns suggest that WNT-5A stimulation leads to the activation of G_i2/3 proteins in N13 cells possibly mediated by FZD₅, the predominant FZD expressed. In summary, we provide for the first time molecular proof that WNT-5A stimulation results in the activation of heterotrimeric G_i2/3 proteins in mammalian cells with physiological protein stochiometry.     

Distribution of the α-subunit of the Guanine Nucleotide-binding Protein Gi2 and its Comparison to Gαo

Abstract

Site specific antisera against a synthetic peptide corresponding to the sequence 3–17 of Gαi2 have been raised and the specificity examined using purified homogeneous G_o, G_i2 and G_i containing a 41 kDa α-subunit. The distribution of Gα_i2 was investigated in plasma membranes from different tissues and cells and compared to the distribution of Gα_o and other pertussis toxin sensitive Gα. Considerable amounts of Gα_io were found in endocrine tissue especially in membranes from the adrenal and thyroid, in leucocytes and platelets where it constitutes the major, if not only, pertussis toxin-sensitive Gα, as well as in some cell lines (C6, NG 108–15, S49 cyc^?); erythrocytes contained a 41 kDa Gα_i which was different from Gα_o. Gα_o was present abundantly in nervous tissue, adrenal medulla and cortex but also found in low amounts in other membranes except for lung, liver and blood cells. Subcellular fractionaltion of cardiac ventricular muscle demonstrated the presence of Gα_o and low amounts of Gα_i2 in sarcolemma, but only 41kDa Gα_i was present in sarcoplasmic reticulum. The importance of the distinct distribution in terms of signal transduction is discussed.     

Thematic Review Series: Lysophospholipids And Their Receptors: LPA receptor signaling: pharmacology,physiology, and pathophysiology

Lysophosphatidic acid (LPA) is a small ubiquitous lipid found in vertebrate and nonvertebrate organisms that mediates diverse biological actions and demonstrates medicinal relevance. LPA’s functional roles are driven by extracellular signaling through at least six 7-transmembrane G protein-coupled receptors. These receptors are named LPA_1–6 and signal through numerous effector pathways activated by heterotrimeric G proteins, including G_i/o, G_12/13, G_q, and G_s. LPA receptor-mediated effects have been described in numerous cell types and model systems, both in vitro and in vivo, through gain- and loss-of-function studies. These studies have revealed physiological and pathophysiological influences on virtually every organ system and developmental stage of an organism. These include the nervous, cardiovascular, reproductive, and pulmonary systems. Disturbances in normal LPA signaling may contribute to a range of diseases, including neurodevelopmental and neuropsychiatric disorders, pain, cardiovascular disease, bone disorders, fibrosis, cancer, infertility, and obesity. These studies underscore the potential of LPA receptor subtypes and related signaling mechanisms to provide novel therapeutic targets.     

Guanine nucleotide binding regulatory proteins in liver from obese humans with and without type II diabetes: Evidence for altered “cross-talk” between the insulin receptor and Gi-proteins

A novel pathway for physiological “cross-talk” between the insulin receptor and the regulatory G_i-protein has been demonstrated. We tested the hypothesis that a coupling defect between G_i and the insulin receptor is present in the liver of obese patients with and without type li diabetes. Insulin 1 × 10^?9 M (～ ED₅₀) and 1 × 10^?7 M (Max) inhibited pertussis toxin-catalyzed ADP ribosylation of G_i in human liver plasma membranes from lean and obese nondiabetic patients. However, 1 × 10^?7 M insulin was without effect in membranes from patients with type II diabetes. This coupling defect was not intrinsic to G_i, since Mg²⁺ and GTPγS inhibited pertussis toxin-catalyzed ADP ribosylation in both diabetic and nondiabetic patients. Binding of insulin of the α-subunit and activation of the tyrosine kinase intrinsic to the β-subunit of the insulin receptor are not responsible for the coupling defect. ¹²⁵I insulin binding is the same in obese patients with or without diabetes. Tyrosine kinase of the insulin receptor is decreased in diabetes. However, a monoclonal antibody to the insulin receptor (MA-20) at equimolar concentrations with insulin equally inhibits pertussis toxin-catalyzed ADP ribosylation of G_i without activating tyrosine kinase or insulin receptor autophosphorylation. Immunodetection of G-proteins suggested that G_i3α was normal in diabetes and G_i1-2α was decreased by 40% in the diabetic group as compared to the obese nondiabetic group but was normal when compared to the lean non diabetic group. We conclude that the novel pathway of insulin signaling involving the regulatory G_i proteins via biochemical mechanisms not directly involving the tyrosine kinase of the insulin receptor is altered in obese type II diabetes and offers a new target for the search of the mechanism(s) of insulin resistance.     

A Region Containing a Proline-Rich Motif Targets sGi2 to the Golgi Apparatus

The central function of heterotrimeric GTP-binding proteins (G proteins) is the transduction of extracellular signals, via membrane receptors, leading to the activation of intracellular effectors. In addition to being associated with the plasma membrane, the α subunits of some of these proteins have also been localized in intracellular compartments. The mRNA of the G-protein inhibitory α subunit 2 (G_αi2) encodes two proteins, G_αi2 and sG_i2, by an alternative splicing mechanism. sG_i2 differs from G_αi2 in the C-terminal region and localizes in the Golgi in contrast to the plasma membrane localization of G_αi2. In this paper we show that the sequence specific to sG_i2 can direct the Golgi localization of other G_αi subunits, but not of the stimulatory subunit G_αs or of a secreted protein. This indicates that, in addition to the sG_i2 C-terminus, sequences located elsewhere in the protein are required to determine the Golgi localization. Inside the sG_i2 C-terminal region we have identified a 14-amino-acid proline-rich motif which specifies the Golgi localization. Finally, we show that the sG_i2 subunit, once activated, leaves the Golgi to be localized in the endoplasmic reticulum.     

Structural Evidence for a Sequential Release Mechanism for Activation of Heterotrimeric G Proteins

Heptahelical G-protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors couple to heterotrimeric G proteins to relay extracellular signals to intracellular signaling networks, but the molecular mechanism underlying guanosine 5′-diphosphate (GDP) release by the G protein α-subunit is not well understood. Amino acid substitutions in the conserved α5 helix of G_i, which extends from the C-terminal region to the nucleotide-binding pocket, cause dramatic increases in basal (receptor-independent) GDP release rates. For example, mutant Gα_i1-T329A shows an 18-fold increase in basal GDP release rate and, when expressed in culture, it causes a significant decrease in forskolin-stimulated cAMP accumulation. The crystal structure of Gα_i1-T329A·GDP shows substantial conformational rearrangement of the switch I region and additional striking alterations of side chains lining the catalytic pocket that disrupt the Mg⁺² coordination sphere and dislodge bound Mg⁺². We propose a “sequential release” mechanism whereby a transient conformational change in the α5 helix alters switch I to induce GDP release. Interestingly, this mechanistic model for heterotrimeric G protein activation is similar to that suggested for the activation of the plant small G protein Rop4 by RopGEF8.     

Characterization of subcellular localization and stability of a splice variant of G alphai2

Background  

Alternative mRNA splicing of α_i2, a heterotrimeric G protein α subunit, has been shown to produce an additional protein, termed sα_i2. In the sα_i2 splice variant, 35 novel amino acids replace the normal C-terminal 24 amino acids of α_i2. Whereas α_i2 is found predominantly at cellular plasma membranes, sα_i2 has been localized to intracellular Golgi membranes, and the unique 35 amino acids of sα_i2 have been suggested to constitute a specific targeting signal.     

Identification and subcellular distribution of the Gi-proteins in the enterocytic-differentiated adenocarcinoma cell-line,Caco-2

Summary— As evidenced by pertussis toxin-catalysed [₃₂P]ADP-ribosylation, immunoblotting and Northern blot, the human adenocarcinoma intestinal cell line Caco-2 expresses G_i2 and G_i3 proteins. The localization of these two G_is within the cell was investigated by using subcellular fractionation and confocal microscopy on intact cell layer. A brush-border rich fraction and a pellet containing the remaining cellular membranes were prepared. [₃₂P]ADP-ribosylation and immunoblotting demonstrated the presence of both α_i2 and α_i3 in these two preparations. Immunofluorescence studies performed on intact cells grown on Transwell filters and viewed by confocal microscopy further confirmed the localization of α_i3-subunit on basolateral as well as on apical membranes. In contrast, α_i2-subunit was shown to accumulate mainly in the intra-cellular compartment while only faint staining of the plasma membrane was detectable. Based upon double-labelling experiments with antibody against rough endoplasmic reticulum (RER), there is a strong possibility that intra-cellular sites of α_i2-subunit correspond to association with RER membranes.     

Diverse β subunits of heterotrimeric G proteins are present in thyroid plasma membranes

The functioning of heterotrimeric G protein α subunits in the transduction of hormonal signals to appropriate intracellular responses is well recognized. Much less is known about the distribution of isoforms and functions of G protein β subunits. Here, using specific antibodies, we documented that in plasma membranes of the thyroid cell line Nthy-ori 3-1 all Gβ isoforms-Gβ₁, Gβ₂, Gβ₃, Gβ₄ and Gβ₅ are present, while the Gβ₃ occurs in minute amount. In plasma membrane fraction isolated from pooled postoperative thyroids of patients with nodular goiter and Graves’ disease, the Gβ₁, Gβ₂, Gβ₄ and Gβ₅ subunits were found, whereas Gβ₃ could not be detected.Competition studies revealed that the Gβ₂ is the principal Gβ subunit in membranes from cultured thyroid cells, originated from normal thyroid, as well as in membranes from patients’ thyroids. This suggests that Gβ₂ subunit cooperates with Gα_s subunit, the most active of the Gα variants, during stimulation of adenylate cyclase which constitutes the main route of physiological thyroid stimulation.     

Expression of the μ-Opioid Receptor in CHO Cells: Ability of μ-Opioid Ligands to Promote α-Azidoanilido[32P]GTP Labeling of Multiple G Protein α Subunits

Abstract: The identities of heterotrimeric G proteins that can interact with the μ-opioid receptor were investigated by α-azidoanilido[³²P]GTP labeling of α subunits in the presence of opioid agonists in Chinese hamster ovary (CHO)-MORIVA3 cells, a CHO clone that stably expressed μ-opioid receptor cDNA (MOR-1). This clone expressed 1.01 × 10⁶μ-opioid receptors per cell and had higher binding affinity and potency to inhibit adenylyl cyclase for the μ-opioid-selective ligands [d -Ala²,N-MePhe⁴,Gly-ol]-enkephalin and [N-MePhe³,d -Pro⁴]-morphiceptin, relative to the δ-selective opioid agonist [d -Pen²,d -Pen⁵]-enkephalin or the κ-selective opioid agonist U-50,488H. μ-Opioid ligands induced an increase in α-azidoanilido[³²P]GTP photoaffinity labeling of four Gα subunits in this clone, three of which were identified as G_i3α, G_i2α, and G_o2α. The same pattern of simultaneous interaction of the μ-opioid receptor with multiple Gα subunits was also observed in two other clones, one expressing about three times more and the other 10-fold fewer receptors as those expressed in CHO-MORIVA3 cells. The opioid-induced increase of labeling of these G proteins was agonist specific, concentration dependent, and blocked by naloxone and by pretreatment of these cells with pertussis toxin. A greater agonist-induced increase of α-azidoanilido[³²P]GTP incorporation into G_i2α (160–280%) and G_o2α (110–220%) than for an unknown Gα (G_?α) (60%) or G_i3α (40%) was produced by three different μ-opioid ligands tested. In addition, slight differences were also found between the ability of various μ-opioid agonists to produce half-maximal labeling (ED₅₀) of any given Gα subunit, with a rank order of G_i3α > G_o2α > G_i2α = G_?α. In any case, these results suggest that the activated μ-opioid receptor couples to four distinct G protein α subunits simultaneously.     

Emerging non-canonical functions for heterotrimeric G proteins in cellular signaling

Abstract

Classically heterotrimeric G proteins have been described as the principal signal transducing machinery for G-protein-coupled receptors. Receptor activation catalyzes nucleotide exchange on the Gα protein, enabling Gα-GTP and Gβγ-subunits to engage intracellular effectors to generate various cellular effects such as second messenger production or regulation of ion channel conductivity. Recent genetic and proteomic screens have identified novel heterotrimeric G-protein-interacting proteins and expanded their functional roles. This review highlights some examples of recently identified interacting proteins and summarizes how they functionally connect heterotrimeric G proteins to previously underappreciated cellular roles.     

A method for the prediction of GPCRs coupling specificity to G-proteins using refined profile Hidden Markov Models

Background  

G- Protein coupled receptors (GPCRs) comprise the largest group of eukaryotic cell surface receptors with great pharmacological interest. A broad range of native ligands interact and activate GPCRs, leading to signal transduction within cells. Most of these responses are mediated through the interaction of GPCRs with heterotrimeric GTP-binding proteins (G-proteins). Due to the information explosion in biological sequence databases, the development of software algorithms that could predict properties of GPCRs is important. Experimental data reported in the literature suggest that heterotrimeric G-proteins interact with parts of the activated receptor at the transmembrane helix-intracellular loop interface. Utilizing this information and membrane topology information, we have developed an intensive exploratory approach to generate a refined library of statistical models (Hidden Markov Models) that predict the coupling preference of GPCRs to heterotrimeric G-proteins. The method predicts the coupling preferences of GPCRs to G_s, G_i/o and G_q/11, but not G_12/13 subfamilies.     

Assembly and Function of the Regulator of G protein Signaling 14 (RGS14)·H-Ras Signaling Complex in Live Cells Are Regulated by Gαi1 and Gαi-linked G Protein-coupled Receptors

Regulator of G protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates heterotrimeric G protein and H-Ras signaling pathways. RGS14 possesses an RGS domain that binds active Gα_i/o-GTP subunits to promote GTP hydrolysis and a G protein regulatory (GPR) motif that selectively binds inactive Gα_i1/3-GDP subunits to form a stable heterodimer at cellular membranes. RGS14 also contains two tandem Ras/Rap binding domains (RBDs) that bind H-Ras. Here we show that RGS14 preferentially binds activated H-Ras-GTP in live cells to enhance H-Ras cellular actions and that this interaction is regulated by inactive Gα_i1-GDP and G protein-coupled receptors (GPCRs). Using bioluminescence resonance energy transfer (BRET) in live cells, we show that RGS14-Luciferase and active H-Ras(G/V)-Venus exhibit a robust BRET signal at the plasma membrane that is markedly enhanced in the presence of inactive Gα_i1-GDP but not active Gα_i1-GTP. Active H-Ras(G/V) interacts with a native RGS14·Gα_i1 complex in brain lysates, and co-expression of RGS14 and Gα_i1 in PC12 cells greatly enhances H-Ras(G/V) stimulatory effects on neurite outgrowth. Stimulation of the Gα_i-linked α_2A-adrenergic receptor induces a conformational change in the Gα_i1·RGS14·H-Ras(G/V) complex that may allow subsequent regulation of the complex by other binding partners. Together, these findings indicate that inactive Gα_i1-GDP enhances the affinity of RGS14 for H-Ras-GTP in live cells, resulting in a ternary signaling complex that is further regulated by GPCRs.     

Atg5-mediated autophagy deficiency in proximal tubules promotes cell cycle G2/M arrest and renal fibrosis

Macroautophagy/autophagy protects against cellular stress. Renal sublethal injury-triggered tubular epithelial cell cycle arrest at G₂/M is associated with interstitial fibrosis. However, the role of autophagy in renal fibrosis is elusive. Here, we hypothesized that autophagy activity in tubular epithelial cells is pivotal for inhibition of cell cycle G₂/M arrest and subsequent fibrogenic response. In both renal epithelial cells stimulated by angiotensin II (AGT II) and the murine kidney after unilateral ureteral obstruction (UUO), we observed that occurrence of autophagy preceded increased production of COL1 (collagen, type I). Pharmacological enhancement of autophagy by rapamycin suppressed COL1 accumulation and renal fibrosis. In contrast, genetic ablation of autophagy by proximal tubular epithelial cell-specific deletion of Atg5, with reduction of the LC3-II protein level and degradation of SQSTM1/p62, showed marked cell cycle arrest at the G₂/M phase, robust COL1 deposition, and severe interstitial fibrosis in a UUO model, as compared with wild-type mice. In vitro, AGT II exposure triggered autophagy preferentially in the G₁/S phase, and increased COL1 expression in the G₂/M phase in renal epithelial cells. Stimulation of Atg5-deficient primary proximal tubular cells with AGT II also resulted in elevated G₂/M arrest and COL1 production. Pharmacological or genetic inhibition of autophagy increased AGT II-mediated G₂/M arrest. Enhanced expression of ATG5, but not the autophagy-deficient ATG5 mutant K130R, rescued the G₂/M arrest, suggesting the regulation of cell cycle progression by ATG5 is autophagy dependent. In conclusion, Atg5-mediated autophagy in proximal epithelial cells is a critical host-defense mechanism that prevents renal fibrosis by blocking G₂/M arrest.     

G Proteins in Reverse Mode: RECEPTOR-MEDIATED GTP RELEASE INHIBITS G PROTEIN AND EFFECTOR FUNCTION*

Active G protein-coupled receptors activate heterotrimeric Gαβγ proteins by catalyzing the exchange of GDP by GTP at the Gα subunit. A paradoxical attenuation of G protein-activated inwardly rectifying potassium channels (GIRK) upon stimulation of native cells with high concentrations of agonist is known. However, a deactivation of activated G proteins by active receptors has not been experimentally studied in intact cells. We monitored GIRK currents and G_o protein activation by means of fluorescence resonance energy transfer (FRET) in parallel. The results suggested that GIRK currents were paradoxically attenuated due to an inactivation of G_o proteins by active α_2A-adrenergic receptors. To study the mechanisms, G protein activation and receptor-G protein interactions were analyzed as a function of nucleotide type and nucleotide concentrations by means of FRET, while controlling intracellular nucleotides upon permeabilization of the cell membrane. Results suggested a receptor-catalyzed dissociation of GTP from activated heterotrimeric Gαβγ. Consequently, nucleotide-free G proteins were sequestrated in heterotrimeric conformation at the active receptor, thus attenuating downstream signaling in an agonist-dependent manner.     

Modified minimal model for effect of physical exercise on insulin sensitivity and glucose effectiveness in type 2 diabetes and healthy human

The Bergman’s minimal model of glucose and insulin plasma levels is commonly used to analyse the results of glucose tolerance tests in humans. In this paper, we present the modified minimal model with plasma insulin compartment under the assumption that if the plasma glucose compartment drops below the basal glucose levels, the rate of insulin entering the plasma glucose compartment is zero. Insulin is cleared from the plasma insulin compartment at a rate proportional to the amount of insulin in the plasma insulin compartment. The modified minimal model was used to study the effect of physical exercise via parameters of a mathematical model to qualitative the magnitude of changes in insulin sensitivity (S _I) and glucose effectiveness (S _G) in response to exercise in type 2 diabetes and healthy human. The short-term effects of physical exercise in type 2 diabetes did not improve S _G, but markedly improved the low S _I values found in type 2 diabetes, indicating that the effects of exercise on S _I are quantitatively important in the interpretation of training-related S _I changes and may even be therapeutically useful in type 2 diabetes patients. Physical exercise is indicated either to prevent or delay the onset of type 2 diabetes or to assure a good control of type 2 diabetes by increasing insulin sensitivity.     

The Peptides Mimicking the Third Intracellular Loop of 5-Hydroxytryptamine Receptors of the Types 1B and 6 Selectively Activate G Proteins and Receptor-Specifically Inhibit Serotonin Signaling via the Adenylyl Cyclase System

The third intracellular loop (ICL3) of G protein-coupled receptors has, as a rule, a key role in their interaction with heterotrimeric G proteins. We synthesized peptides corresponding to the C-terminal region of the ICL3 (C-ICL3) of 5-hydroxytryptamine receptors of the type 1B (5-HT_1BR) and 6 (5-HT₆R) and studied their influence on the functional activity of adenylyl cyclase signaling system (ACSS) in synaptosomal membranes isolated from the rat brain. The 5-HT_1BR-peptide ARERKATKTL^307–316K-amide mimicking agonist-activated 5-HT_1BR reduced forskolin-stimulated adenylyl cyclase (AC) activity and activated pertussis toxin-sensitive G proteins. It lowered inhibitory effects of serotonin and 5-HT_1BR-agonists on forskolin-stimulated AC activity and their stimulating effects on GTP binding. This was not the case in the presence of 5-HT_1BR-antagonists. The 5-HT₆R-peptides mimicking 5-HT₆R activated both the basal AC activity and GTP binding of cholera toxin-sensitive G proteins. They lowered the stimulating effect of serotonin and 5-HT₆R-agonists on AC and G_s proteins, but in the presence of 5-HT₆R-antagonists their action was blocked. Of all the 5-HT₆R-peptides with linear and dimeric structure we studied the palmitoylated peptide KHSRKALKASL^258–268K(Pal)A-amide had a most pronounced effect both on the basal and 5-HT₆R-agonist-stimulated ACSS. The data was obtained indicating that the peptides corresponding to C-ICL3 of 5-HT_1BR and 5-HT₆R selectively activate G_i and G_s proteins, respectively, and in a receptor-specific manner reduce signal transduction via serotonin-sensitive ACSS in the rat brain. The results of the study give strong evidence in favor of active participation of C-ICL3 of these 5-HTRs in their coupling with the G proteins.