Gi Subtypes Research Articles

Applying GC-MS based serum metabolomic profiling to characterize two traditional Chinese medicine subtypes of diabetic foot gangrene.

Traditional Chinese medicine (TCM) has a long history and particular advantages in the diagnosis and treatment of diabetic foot gangrene (DFG). Patients with DFG are mainly divided into two subtypes, tendon lesion with edema (GT) and ischemic lesion without edema (GI), which are suitable for different medical strategies. Metabolomics has special significance in unravelling the complexities of multifactorial and multisystemic disorders. This study acquired the serum metabolomic profiles of two traditional Chinese medicine subtypes of DFG to explore potential molecular evidence for subtype characterization, which may contribute to the personalized treatment of DFG. A total of 70 participants were recruited, including 20 with DM and 50 with DFG (20 with GI and 30 with GT). Conventional gas chromatography-mass spectrometry (GC-MS) followed by orthogonal partial least-squares discriminant analysis (OPLS-DA) were used as untargeted metabolomics approaches to explore the serum metabolomic profiles. Kyoto encyclopedia of genes and genomes (KEGG) and MetaboAnalyst were used to identify the related metabolic pathways. Compared with DM patients, the levels of 14 metabolites were altered in the DFG group, which were also belonged to the differential metabolites of GI (13) and GT (7) subtypes, respectively. Among these, urea, α-D-mannose, cadaverine, glutamine, L-asparagine, D-gluconic acid, and indole could be regarded as specific potential metabolic markers for GI, as well as L-leucine for GT. In the GI subtype, D-gluconic acid and L-asparagine are positively correlated with activated partial thromboplastin time (APTT) and fibrinogen (FIB). In the GT subtype, L-leucine is positively correlated with the inflammatory marker C-reactive protein (CRP). Arginine and proline metabolism, glycine, serine and threonine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis are the most important metabolic pathways associated with GI. The main metabolic pathways related to GT include pyrimidine metabolism, glutathione metabolism, biosynthesis of valine, leucine, and isoleucine, as well as valine, serine, and isoleucine with metabolites. The results of this study indicate that patients with different DFG subtypes have distinct metabolic profiles, which reflect the pathological characteristics of each subtype respectively. These findings will help us explore therapeutic targets for DFG and develop precise treatment strategies.

Structure-based design of bitopic ligands for the µ-opioid receptor.

Mu-opioid receptor (µOR) agonists such as fentanyl have long been used for pain management, but are considered a major public health concern owing to their adverse side effects, including lethal overdose1. Here, in an effort to design safer therapeutic agents, we report an approach targeting a conserved sodium ion-binding site2 found in µOR3 and many other class A G-protein-coupled receptors with bitopic fentanyl derivatives that are functionalized via a linker with a positively charged guanidino group. Cryo-electron microscopy structures of the most potent bitopic ligands in complex with µOR highlight the key interactions between the guanidine of the ligands and the key Asp2.50 residue in the Na+ site. Two bitopics (C5 and C6 guano) maintain nanomolar potency and high efficacy at Gi subtypes and show strongly reduced arrestin recruitment-one(C6 guano) also shows the lowest Gz efficacy among the panel of µOR agonists, including partial and biased morphinan and fentanyl analogues. In mice, C6 guano displayed µOR-dependent antinociception with attenuated adverse effects, supporting the µOR sodium ion-binding site as a potential target for the design of safer analgesics. In general, our study suggests that bitopic ligands that engage the sodium ion-binding pocket in class A G-protein-coupled receptors can be designed to control their efficacy and functional selectivity profiles for Gi, Go and Gz subtypes and arrestins, thus modulating their in vivo pharmacology.

Functional Selectivity at Cannabinoid Receptors.

It is now clear that, in contrast to traditional descriptions of G protein-coupled receptor signaling, agonists can activate or inhibit characteristic patterns of downstream effector pathways depending on their structures and the conformational changes induced in the receptor. This is referred to as functional selectivity (also known as agonist-directed trafficking, ligand-induced differential signaling, or biased agonism). It is important because even small structural differences can result in significant variations in overall agonist effects (wanted and unwanted) depending on which postreceptor signaling systems are engaged by each agonist/receptor pairing. In addition to the canonical signaling pathways mediated by Gi/o proteins, CB1 and CB2 receptor agonists can have effects via differential activation not only of Gi subtypes but also of Gs and Gq/11 proteins. For example, the classical cannabinoid HU-210 produces maximal activation of both Gi and Go proteins, while the endocannabinoid anandamide and aminoalkylindole WIN 55,212 both produce maximal activation of Gi, but submaximal activation of Go. Cannabinoid agonists can also signal differentially via β-arrestins coupled to mitogen-activated protein kinases, subsequently promoting varying degrees of receptor internalization and agonist desensitization. A recent extensive characterization of the molecular pharmacology of CB2 agonists (Soethoudt et al., 2017) identified marked differences (bias) in the ability of certain agonists to activate distinct signaling pathways (cAMP accumulation, ERK phosphorylation, GIRK activation, GTPγS binding, and β-arrestin recruitment) and to cause off-target effects, exemplifying the need to evaluate functional selectivity in agonist drug development.

Detection and typing of caliciviruses from patients with acute diarrhea in Hangzhou area, 2009 - 2010

To investigate the molecular-epidemiologic characteristics and genotypes of human calicivirus (HuCVs) in acute diarrhea patients in Hangzhou from 2009 to 2010. Epidemiologic data and fecal specimens were collected from patients with acute diarrhea. HuCVs of 920 specimens were detected by PCR. PCR products of several positive samples were randomly selected and sequenced. All the sequences were analyzed, phylogenetically. 201 HuCVs positive cases were identified from 920 facal specimens (21.8%). 25 isolates would include norovirus GI-type, GII-type for 170 strains and sapovirus for 11 strains. Norovirus GI-type and GII-type were detected in four specimens at the same time. Other specimens were mixed infection with norovirus GII-type and sapovirus. Genotypes of HuCVs showed that norovirus GI subtypes were GI-1 (3 strains) and GI-2 (1 strain). Norovirus GII subtypes were GII-4/2006b variant strains (7 strains), GII-2 (1 strain), GII-7 (1 strain) and GII-4/2008 variant strains (2 strains); Sapovirus subtypes were GI-2 (5 strains), GI-1 (4 strains) and GII-1 (1 strain). The prevalence rates of HuCVs were different in seasons and age groups. HuCVs were one of the major pathogens causing acute diarrhea. Both multiple viruses and genotypes of HuCVs were found in the specimens. GII-4/2006b variant and similar strains were identified, probably as the prevalent strains from 2009 to 2010 in Hangzhou, Zhejiang province.

Chemically Distinct Ligands Promote Differential CB1Cannabinoid Receptor-Gi Protein Interactions

To understand how structurally distinct ligands regulate CB(1) receptor interactions with Gi1, Gi2, and Gi3, we quantified the Galphai and betagamma proteins that coimmunoprecipitate with the CB(1) receptor from a detergent extract of N18TG2 membranes in the presence of ligands. A mixture of A, R, G(GDP) (or G_), and ARG(GDP) (or ARG_) complexes was observed in the presence of aminoalkylindole (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN 55,212-2) for all three RGalphai complexes, cannabinoid desacetyllevonantradol for Galphai1 and Galphai2, and eicosanoid (R)-methanandamide for Galphai3. Desacetyllevonantradol maintained RGalphai3 complexes and (R)-methanandamide maintained RGalphai1 and RGalphai2 complexes even in the presence of a nonhydrolyzable GTP analog. The biaryl pyrazole antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR141716) maintained all three RGalphai complexes. Gbeta proteins, and to a certain extent Ggamma2, exhibited the same association/dissociation pattern as the Galpha proteins. A GDP analog had no influence on any of these association/dissociation reactions and failed to promote sequestration of G proteins. These results can be explained by invoking the existence of an inverse agonist-supported inactive state in the ternary complex equilibrium model. WIN 55,212-2 behaves as an agonist for all three Gi subtypes; SR141716 behaves as an inverse agonist for all three Gi subtypes; desacetyllevonantradol behaves as an agonist for Gi1 and Gi2, and an inverse agonist at Gi3; and (R)-methanandamide behaves as an inverse agonist at Gi1 and Gi2, and an agonist at Gi3. These ligand-selective G protein responses imply that multiple conformations of the receptor could be evoked by ligands to regulate individual G proteins.

CB1 receptor-G protein association. Subtype selectivity is determined by distinct intracellular domains.

The CB1 cannabinoid receptor in N18TG2 neuroblastoma cells inhibits adenylate cyclase, and this response can be mimicked by a peptide corresponding to the juxtamembrane C-terminal domain (CB(1)401-417). Guanosine 5'-O-(3-thio)triphosphate binding to G proteins can be stimulated by both peptide CB(1)401-417 and peptides corresponding to the third intracellular loop [Howlett, A.C., Song, C., Berglund, B.A., Wilken, G.H. & Pigg, J.J. (1998) Mol. Pharmacol. 53, 504-510; Mukhopadhyay, S., Cowsik, S.M., Welsh, W.J. & Howlett, A.C. (1999) Biochemistry 38, 3447-3455]. In Chaps-solubilized N18TG2 membranes, the CB1 receptor coimmunoprecipitated with all three Gi subtypes. Pertussis toxin significantly reduced the CB(1) receptor-G alpha(i) association and attenuated the CB(1)401-417-induced inhibition of adenylate cyclase. CB(1)401-417 significantly reduced the CB(1) receptor association with G alpha(i3), but not with G alpha(i1) or G alpha(i2). In contrast, third intracellular loop peptides significantly reduced the CB(1) receptor association with G alpha(i1) and G alpha(i2), but not G alpha(i3). These interactions are specific for the CB(1) receptor because a peptide corresponding to the juxtamembrane C-terminal domain of the CB(2) receptor failed to compete for the association of the CB1 receptor with any of the Gi alpha subtypes, and was not able to activate Gi proteins to inhibit adenylate cyclase. These studies indicate that different domains of the CB(1) receptor direct the interaction with specific G protein subtypes.

Tumor Necrosis Factor α Stimulates Lipolysis in Adipocytes by Decreasing Gi Protein Concentrations

Prolonged treatment (12-24 h) of adipocytes with tumor necrosis factor alpha (TNFalpha) stimulates lipolysis. We have investigated the hypothesis that TNFalpha stimulates lipolysis by blocking the action of endogenous adenosine. Adipocytes were incubated for 48 h with TNFalpha, and lipolysis was measured in the absence or presence of adenosine deaminase. Without adenosine deaminase, the rate of glycerol release was 2-3-fold higher in the TNFalpha-treated cells, but with adenosine deaminase lipolysis increased in the controls to approximately that in the TNFalpha-treated cells. This suggests that TNFalpha blocks adenosine release or prevents its antilipolytic effect. Both N6-phenylisopropyl adenosine and nicotinic acid were less potent and efficacious inhibitors of lipolysis in treated cells. A decrease in the concentration of alpha-subunits of all three Gi subtypes was detected by Western blotting without a change in Gs proteins or beta-subunits. Gi2alpha was about 50% of control, whereas Gi1alpha and Gi3alpha were about 20 and 40% of control values, respectively. The time course of Gi down-regulation correlated with the stimulation of lipolysis. Furthermore, down-regulation of Gi by an alternative approach (prolonged incubation with N6-phenylisopropyl adenosine) stimulated lipolysis. These findings indicate that TNFalpha stimulates lipolysis by blunting endogenous inhibition of lipolysis. The mechanism appears to be a Gi protein down-regulation.

Insulin resistance in adipocytes after downregulation of Gi subtypes.

To determine whether downregulation of Gi proteins is associated with insulin resistance, we incubated isolated adipocytes with N6-(2-phenylisopropyl)adenosine (PIA; an A1-adenosine receptor agonist; 300 nM), prostaglandin E1 (PGE1; 3 microM), or nicotinic acid (1 mM) for 4 days in primary culture. The cells were washed, and the rate of glucose transport (2-deoxy-[3H]glucose uptake) was measured after incubation with various concentrations of insulin for 45 min. Both PIA and PGE1 (which downregulate Gi) decreased the maximal responsiveness of the cells to insulin by approximately 30% and caused a rightward shift in the dose-response curve. By contrast, nicotinic acid (which does not downregulate Gi) did not alter the insulin sensitivity of the cells. Prolonged treatment of adipocytes with either PIA or PGE1 (but not nicotinic acid) rendered the cells completely resistant to the antilipolytic effect of insulin. The ability of insulin to stimulate autophosphorylation of the beta-subunit of the insulin receptor was decreased by approximately 30% in PIA-treated cells, and the dose-response curve was shifted to the right. Similarly, the ability of the receptor to phosphorylate poly(Glu4-Tyr1) was decreased by approximately 35%. This decrease in tyrosine kinase activity of the receptor may account for the decrease in insulin sensitivity of glucose transport but cannot account for the complete loss of antilipolysis. The findings suggest both a direct and indirect involvement of Gi proteins in insulin action.

Selective activation of Gi1 and Gi2 in mouse sperm by the zona pellucida, the egg's extracellular matrix.

Mammalian sperm acrosomal exocytosis as induced by the egg's zona pellucida (ZP) appears to be a G protein-mediated event. Previously, we demonstrated that ZP3, the acrosome reaction-inducing component of mouse ZP, activates one or more Gi subtypes in isolated sperm membranes (Ward, C. R., Storey, B. T., and Kopf, G. S. (1992) J. Biol. Chem. 267, 14061-14067). To determine the identity of the Gi subtype(s) activated in the sperm, we examined ligand-dependent decreases in pertussis toxin-catalyzed in vitro [32P]ADP-ribosylation of the different Gi alpha subtypes that were identified by immunoprecipitation using Gi alpha-specific antisera. Membranes treated with solubilized ZP or mastoparan and subsequently [32P]ADP-ribosylated using pertussis toxin displayed 35 and 56% decreases, respectively, in [32P]ADP-ribosylation when compared with controls. These changes were quantitatively similar to the percentage increases in 35S-labeled guanosine 5'-O-(thiotriphosphate) binding in membranes treated with equivalent concentrations of ZP or mastoparan (Ward et al., 1992). Immunoprecipitation with Gi alpha subtype-specific antisera and subsequent autoradiography revealed that mastoparan activated Gi1, Gi2, and Gi3 in the membranes. ZP, in contrast, selectively activated Gi1 and Gi2. These data demonstrate that the egg's extracellular matrix has the ability to activate selectively sperm membrane Gi subtypes.

Stimulation of human thyroid growth via the inhibitory guanine nucleotide binding (G) protein Gi: constitutive expression of the G-protein alpha subunit Gi alpha-1 in autonomous adenoma.

The alpha subunits of the stimulatory and inhibitory G proteins, Gs alpha and Gi alpha, activate transmembrane-signaling systems involved in the control of cell proliferation. We have investigated the pattern of expression of Gi alpha subtypes and Gi alpha-mediated proliferative responses in the human thyroid. Human thyroid membranes contain two subtypes of Gi alpha, Gi alpha-1 and Gi alpha-2, as assessed by using specific antibodies. The expression of Gi alpha-1 is under tight control by thyrotropin in vivo and in primary cultures of thyroid epithelial cells. In contrast, Gi alpha-1 is expressed in the absence of thyrotropin in thyroid autonomous adenoma, an endocrine-active tumor, and its levels are not regulated by thyrotropin in thyroid epithelial cells prepared from these tumors. If thyroid epithelial cells are treated with pertussis toxin to block signal transduction via Gi, the mitogenic response to serum factors is reduced. These observations demonstrate that Gi subtypes transmit growth stimuli in the human thyroid. The constitutive expression of Gi alpha-1 in autonomous adenoma may allow for the unregulated stimulation of thyroid cell proliferation by a yet unidentified signaling pathway and, thus, be causally related to autonomous growth of thyroid cells.

Gi down-regulation as a mechanism for heterologous desensitization in adipocytes.

Prolonged incubation of rat adipocytes with (-)N6-phenylisopropyl adenosine (PIA) (an A1 adenosine receptor agonist) leads to down-regulation of each of the three subtypes of Gi (Green, A., Johnson, J. L., and Milligan, G. (1990) J. Biol. Chem. 265, 5206-5210). To determine whether other inhibitors of adenylylcyclase would have similar actions, we incubated adipocytes in primary culture with PIA, prostaglandin E1 (PGE1), or nicotinic acid. After various times cells were homogenized, and crude membrane fractions were analyzed on Western blots using antipeptide antisera to alpha- and beta-subunits of G-proteins (SG1 (which binds to alpha i1 and alpha i2), I3B (which binds to alpha i3), BN2 (binds to beta-subunits) and CS1 (recognizes forms of alpha s)). PIA and PGE1 caused approximately 90% down-regulation of alpha i1 and alpha i3, and about 50% loss of alpha i2 and beta-subunits. In contrast, nicotinic acid at concentrations up to 1 mM had no effect on levels of any of these Gi subtypes. None of the compounds altered levels of either a 43- or 47-kDa form of alpha s. PIA caused about a 50% decrease in binding of [3H]DPCPX (an A1 adenosine receptor antagonist), indicating adenosine receptor down-regulation; however, neither PGE1 nor nicotinic acid treatment altered [3H]DPCPX binding. None of the treatments affected the activity of adenylylcyclase when measured in the presence of 100 microM forskolin and 10 mM Mn2+, indicating that the catalytic subunit of adenylylcyclase is not altered. To determine whether Gi down-regulation results in heterologous desensitization, we incubated adipocytes with maximally effective concentrations of PIA (300 nM), PGE1 (3 microM), or nicotinic acid (1 mM) for 4 days. The cells were then washed and incubated for an additional 30 min with various concentrations of these compounds to determine their ability to inhibit lipolysis. PIA caused a (marked) decrease in the sensitivity of the cells to both PIA and PGE1, thus indicating heterologous desensitization. Similarly, PGE1 decreased the sensitivity of the cells to both PGE1 and PIA, again demonstrating heterologous desensitization. In contrast, prolonged incubation with nicotinic acid decreased the sensitivity of the cells to nicotinic acid but had no effect on the sensitivity of the cells to PIA. Adenylylcyclase in membranes from PGE1-treated cells showed decreased sensitivity to inhibition by PIA. In contrast, adenylylcyclase showed normal sensitivity to PIA in membranes from nicotinic acid-treated cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Down-regulation of the α-subunits of Gi subtypes by prolonged incubation of adipocytes with N6-phenylisopropyl adenosine

Down-regulation of the α-subunits of Gi subtypes by prolonged incubation of adipocytes with <i>N</i>6-phenylisopropyl adenosine

Specificity of receptor-G protein interactions. Discrimination of Gi subtypes by the D2 dopamine receptor in a reconstituted system.

The selectivity of D2 dopamine receptor-guanine nucleotide-binding protein (G protein) coupling was studied by reconstitution techniques utilizing purified D2 dopamine receptors from bovine anterior pituitary and resolved G proteins from bovine brain, bovine pituitary, and human erythrocyte. Titration of a fixed receptor concentration with varying G protein concentrations revealed two aspects of receptor-G protein coupling. First, Gi2 appeared to couple selectively with the D2 receptor with approximately 10-fold higher affinity than any other tested Gi subtype. Second, the G proteins differed in the maximal receptor-mediated agonist stimulation of the intrinsic GTPase activity. Gi2 appeared to be maximally stimulated by agonist-receptor complex with turnover numbers of approximately 2 min-1. The other Gi subtypes, Gi1 and Gi3, could be only partially activated, resulting in maximal rates of GTPase of approximately 1 min-1. Agonist-stimulated GTPase activity was not detected in preparations containing Go from bovine brain. The differences in maximal agonist-stimulated GTPase rates observed among the Gi subtypes could be explained by differences in agonist-promoted guanyl nucleotide exchange. Both guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding and GDP release parameters were enhanced 2-fold for the Gi2 subtype over the other Gi subtypes. These results suggest that even though several types of pertussis toxin substrate may exist in most tissues, a receptor may interact discretely with G proteins, thereby dictating signal transduction mechanisms.

Down-regulation of Gi sub-types by prolonged incubation of adipocytes with an A1 adenosine receptor agonist.

We have reported previously that prolonged incubation of adipocytes with (-)-N6-phenylisopropyl adenosine (PIA) (an A1 adenosine receptor agonist) down-regulates A1 adenosine receptors. There was a concomitant decrease in pertussis toxin catalyzed ADP-ribosylation of a 41-kDa peptide thought to be the alpha-subunit of Gi. To determine whether this represents true down-regulation of the G-protein, and if so which of the three known forms of Gi are down-regulated, we have used antipeptide antisera specific for Gi alpha-subunits. Serum SG1 recognizes alpha i1 and -2, I1C recognizes only alpha i1, and I3B recognizes alpha i3. Rat adipocytes were maintained in primary culture for up to 7 days with 0-1000 nM PIA. Crude membrane preparations were analyzed by Western blots. There was almost complete loss of alpha i1 and -3, and about 50% loss of alpha i2 from PIA-treated cells. The loss of each alpha i was detectable after 24 h with 300 nM PIA and maximal by 4 days. After 4 days, down-regulation was detectable with 3 nM and maximal with 100 nM PIA. Antiserum BN2 demonstrated approximately 50% loss of G-protein beta-subunits in cells treated with 300 nM PIA for 4 days. When cells were incubated for 4 days with 300 nM PIA and then washed to remove PIA, alpha i1, -2, and -3 and beta-subunits returned to control levels within 5 days. Antiserum CS1 detected normal amounts of both the 43- and 47-kDa forms of Gs alpha in PIA-treated cells. We conclude that Gi alpha-subunits are down-regulated along with the adenosine receptor in rat adipocytes.