Two distinct aspects of coupling between Gα(i) protein and G protein-activated K+ channel (GIRK) revealed by fluorescently labeled Gα(i3) protein subunits

G protein-activated K(+) channels (Kir3 or GIRK) are activated by direct interaction with Gβγ. Gα is essential for specific signaling and regulates basal activity of GIRK (I(basal)) and kinetics of the response elicited by activation by G protein-coupled receptors (I(evoked)). These regulations are believed to occur within a GIRK-Gα-Gβγ signaling complex. Fluorescent energy resonance transfer (FRET) studies showed strong GIRK-Gβγ interactions but yielded controversial results regarding the GIRK-Gα(i/o) interaction. We investigated the mechanisms of regulation of GIRK by Gα(i/o) using wild-type Gα(i3) (Gα(i3)WT) and Gα(i3) labeled at three different positions with fluorescent proteins, CFP or YFP (xFP). Gα(i3)xFP proteins bound the cytosolic domain of GIRK1 and interacted with Gβγ in a guanine nucleotide-dependent manner. However, only an N-terminally labeled, myristoylated Gα(i3)xFP (Gα(i3)NT) closely mimicked all aspects of Gα(i3)WT regulation except for a weaker regulation of I(basal). Gα(i3) labeled with YFP within the Gα helical domain preserved regulation of I(basal) but failed to restore fast I(evoked). Titrated expression of Gα(i3)NT and Gα(i3)WT confirmed that regulation of I(basal) and of the kinetics of I(evoked) of GIRK1/2 are independent functions of Gα(i). FRET and direct biochemical measurements indicated much stronger interaction between GIRK1 and Gβγ than between GIRK1 and Gα(i3). Thus, Gα(i/o)βγ heterotrimer may be attached to GIRK primarily via Gβγ within the signaling complex. Our findings support the notion that Gα(i/o) actively regulates GIRK. Although regulation of I(basal) is a function of Gα(i)(GDP), our new findings indicate that regulation of kinetics of I(evoked) is mediated by Gα(i)(GTP).     

Development of inhibitors of heterotrimeric Gαi subunits

Heterotrimeric G-proteins are the immediate downstream effectors of G-protein coupled receptors (GPCRs). Endogenous protein guanine nucleotide dissociation inhibitors (GDIs) like AGS3/4 and RGS12/14 function through GPR/Goloco GDI domains. Extensive characterization of GPR domain peptides indicate they function as selective GDIs for Gα_i by competing for the GPCR and Gβγ and preventing GDP release. We modified a GPR consensus peptide by testing FGF and TAT leader sequences to make the peptide cell permeable. FGF modification inhibited GDI activity while TAT preserved GDI activity. TAT-GPR suppresses G-protein coupling to the receptor and completely blocked α₂-adrenoceptor (α₂AR) mediated decreases in cAMP in HEK293 cells at 100 nM. We then sought to discover selective small molecule inhibitors for Gα_i. Molecular docking was used to identify potential molecules that bind to and stabilize the Gα_i–GDP complex by directly interacting with both Gα_i and GDP. Gα_i–GTP and Gα_q–GDP were used as a computational counter screen and Gα_q–GDP was used as a biological counter screen. Thirty-seven molecules were tested using nucleotide exchange. STD NMR assays with compound 0990, a quinazoline derivative, showed direct interaction with Gα_i. Several compounds showed Gα_i specific inhibition and were able to block α₂AR mediated regulation of cAMP. In addition to being a pharmacologic tool, GDI inhibition of Gα subunits has the advantage of circumventing the upstream component of GPCR-related signaling in cases of overstimulation by agonists, mutations, polymorphisms, and expression-related defects often seen in disease.     

Characterization of heterotrimeric nucleotide-depleted Gα(i)-proteins by Bodipy-FL-GTPγS fluorescence anisotropy

Recombinant heterotrimeric G-protein α_i1, α_i2 and α_i3 subunits were purified in GDP-depleting conditions by affinity chromatography using StrepII-tagged β₁γ₂ subunits. Real-time monitoring of fluorescence anisotropy of Bodipy-FL-GTPγS was used for characterization of nucleotide binding properties and inactivation of the purified proteins. All GDP-depleted α_i were unstable at room temperature and therefore nucleotide binding could be characterized only in a nonequilibrium state. In comparison to Mg²⁺, Mn²⁺ inhibited nucleotide binding to all α_i-heterotrimers studied and accelerated nucleotide release. Mn²⁺ had stabilizing effect on the nucleotide free state of the α_i1 subunit, whereas both Mn²⁺ as well as G-protein activation by mastoparan destabilized the α_i2 subunit.     

The influence of monovalent cations on trimeric G protein G(i)1α activity in HEK293 cells stably expressing DOR-G(i)1α (Cys(351)-Ile(351)) fusion protein

The effect of monovalent cations on trimeric G protein G(i)1α was measured at equimolar concentration of chloride anion in pertussis-toxin (PTX)-treated HEK293 cells stably expressing PTX-insensitive DOR- G(i)1α (Cys(351)-Ile(351)) fusion protein by high-affinity [(35)S]GTPgammaS binding assay. The high basal level of binding was detected in absence of DOR agonist and monovalent ions and this high level was inhibited with the order of: Na(+) > K(+) > Li(+). The first significant inhibition was detected at 1 mM NaCl. The inhibition by monovalent ions was reversed by increasing concentrations of DOR agonist DADLE. The maximum DADLE response was also highest for sodium and decreased in the order of: Na(+) > K(+) ~ Li(+). Our data indicate i) an inherently high activity of trimeric G protein G(i)1α when expressed within DOR- G(i)1α fusion protein and determined in the absence of monovalent cations, ii) preferential sensitivity of DOR- G(i)1alpha to sodium as far as maximum of agonist response is involved.     

G��i and G�¦� Jointly Regulate the Conformations of a G�¦� Effector, the Neuronal G Protein-activated K+ Channel (GIRK)

Stable complexes among G proteins and effectors are an emerging concept in cell signaling. The prototypical Gβγ effector G protein-activated K⁺ channel (GIRK; Kir3) physically interacts with Gβγ but also with Gα_i/o. Whether and how Gα_i/o subunits regulate GIRK in vivo is unclear. We studied triple interactions among GIRK subunits 1 and 2, Gα_i3 and Gβγ. We used in vitro protein interaction assays and in vivo intramolecular Förster resonance energy transfer (i-FRET) between fluorophores attached to N and C termini of either GIRK1 or GIRK2 subunit. We demonstrate, for the first time, that Gβγ and Gα_i3 distinctly and interdependently alter the conformational states of the heterotetrameric GIRK1/2 channel. Biochemical experiments show that Gβγ greatly enhances the binding of GIRK1 subunit to Gα_i3^GDP and, unexpectedly, to Gα_i3^GTP. i-FRET showed that both Gα_i3 and Gβγ induced distinct conformational changes in GIRK1 and GIRK2. Moreover, GIRK1 and GIRK2 subunits assumed unique, distinct conformations when coexpressed with a “constitutively active” Gα_i3 mutant and Gβγ together. These conformations differ from those assumed by GIRK1 or GIRK2 after separate coexpression of either Gα_i3 or Gβγ. Both biochemical and i-FRET data suggest that GIRK acts as the nucleator of the GIRK-Gα-Gβγ signaling complex and mediates allosteric interactions between Gα_i^GTP and Gβγ. Our findings imply that Gα_i/o and the Gα_iβγ heterotrimer can regulate a Gβγ effector both before and after activation by neurotransmitters.     

Integration of G Protein α (Gα) Signaling by the Regulator of G Protein Signaling 14 (RGS14)

RGS14 contains distinct binding sites for both active (GTP-bound) and inactive (GDP-bound) forms of Gα subunits. The N-terminal regulator of G protein signaling (RGS) domain binds active Gα_i/o-GTP, whereas the C-terminal G protein regulatory (GPR) motif binds inactive Gα_i1/3-GDP. The molecular basis for how RGS14 binds different activation states of Gα proteins to integrate G protein signaling is unknown. Here we explored the intramolecular communication between the GPR motif and the RGS domain upon G protein binding and examined whether RGS14 can functionally interact with two distinct forms of Gα subunits simultaneously. Using complementary cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive Gα_i1-GDP at the plasma membrane and that free cytosolic RGS14 is recruited to the plasma membrane by activated Gα_o-AlF₄⁻. Bioluminescence resonance energy transfer studies showed that RGS14 adopts different conformations in live cells when bound to Gα in different activation states. Hydrogen/deuterium exchange mass spectrometry revealed that RGS14 is a very dynamic protein that undergoes allosteric conformational changes when inactive Gα_i1-GDP binds the GPR motif. Pure RGS14 forms a ternary complex with Gα_o-AlF₄⁻ and an AlF₄⁻-insensitive mutant (G42R) of Gα_i1-GDP, as observed by size exclusion chromatography and differential hydrogen/deuterium exchange. Finally, a preformed RGS14·Gα_i1-GDP complex exhibits full capacity to stimulate the GTPase activity of Gα_o-GTP, demonstrating that RGS14 can functionally engage two distinct forms of Gα subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals in host CA2 hippocampal neurons to modulate synaptic plasticity.     

D(2)-like dopamine and β-adrenergic receptors form a signaling complex that integrates G(s)- and G(i)-mediated regulation of adenylyl cyclase

β-Adrenergic receptors (βAR) and D(2)-like dopamine receptors (which include D(2)-, D(3)- and D(4)-dopamine receptors) activate G(s) and G(i), the stimulatory and inhibitory heterotrimeric G proteins, respectively, which in turn regulate the activity of adenylyl cyclase (AC). β(2)-Adrenergic receptors (β(2)AR) and D(4)-dopamine receptors (D(4)DR) co-immunoprecipitated when co-expressed in HEK 293 cells, suggesting the existence of a signaling complex containing both receptors. In order to determine if these receptors are closely associated with each other, and with other components involved in G protein-mediated signal transduction, β(2)AR, D(4)DR, G protein subunits (Gα(i1) and the Gβ(1)γ(2) heterodimer) and AC were tagged so that bioluminescence resonance energy transfer (BRET) could be used to monitor their interactions. All of the tagged proteins retained biological function. For the first time, FlAsH-labeled proteins were used in BRET experiments as fluorescent acceptors for the energy transferred from Renilla luciferase-tagged donor proteins. Our experiments revealed that β(2)AR, D(4)DR, G proteins and AC were closely associated in a functional signaling complex in cellulo. Furthermore, BRET experiments indicated that although activation of G(i) caused a conformational change within the heterotrimeric protein, it did not cause the Gβγ heterodimer to dissociate from the Gα(i1) subunit. Evidence for the presence of a signaling complex in vivo was obtained by purifying βAR from detergent extracts of mouse brain with alprenolol-Sepharose and showing that the precipitate also contained both D(2)-like dopamine receptors and AC.     

Rig-I^-/- mice develop colitis associated with downregulation of Gαi2

RIG-I （retinoid acid-inducible gene-I）, a putative RNA helicase with a cytoplasmic caspase-recrultment domain （CARD）, was identified as a pattem-recognition receptor （PRR） that mediates antiviral immunity by inducing type I interferon production. To further study the biological function of RIG-I, we generated Rig-I^-/- mice through homologous recombination, taking a different strategy to the previously reported strategy. Our Rig-I^-/- mice are viable and fertile. Histological analysis shows that Rig-I^-/ mice develop a colitis-like phenotype and increased susceptibility to dextran sulfate sodium-induced colitis. Accordingly, the size and number of Peyer＇s patches dramatically decreased in mutant mice. The peripheral T-cell subsets in mutant mice are characterized by an increase in effector T cells and a decrease in naive T cells, indicating an important role for Rig-I in the regulation ofT-cell activation. It was further found that Rig-I deficiency leads to the downregulation of G protein αi2 subunit （Gαi2） in various tissues, including T and B lymphocytes. By contrast, upregulation of Rig-I in NB4 cells that are treated with ATRA is accompanied by elevated Gαi2 expression. Moreover, Gαi2 promoter activity is increased in co-transfected NIH3T3 cells in a Rig-I dose-dependent manner. All these findings suggest that Rig-I has crucial roles in the regulation of Gαi2 expression and T-cell activation. The development of colitis may be, at least in part, associated with downregulation of Gαi2 and disturbed T-cell homeostasis.     

Gαi2- and Gαi3-specific regulation of voltage-dependent L-type calcium channels in cardiomyocytes

Background

Two pertussis toxin sensitive G_i proteins, G_i2 and G_i3, are expressed in cardiomyocytes and upregulated in heart failure. It has been proposed that the highly homologous G_i isoforms are functionally distinct. To test for isoform-specific functions of G_i proteins, we examined their role in the regulation of cardiac L-type voltage-dependent calcium channels (L-VDCC).

Methods

Ventricular tissues and isolated myocytes were obtained from mice with targeted deletion of either Gα_i2 (Gα_i2 ^−/−) or Gα_i3 (Gα_i3 ^−/−). mRNA levels of Gα_i/o isoforms and L-VDCC subunits were quantified by real-time PCR. Gα_i and Ca_vα₁ protein levels as well as protein kinase B/Akt and extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation levels were assessed by immunoblot analysis. L-VDCC function was assessed by whole-cell and single-channel current recordings.

Results

In cardiac tissue from Gα_i2 ^−/− mice, Gα_i3 mRNA and protein expression was upregulated to 187±21% and 567±59%, respectively. In Gα_i3 ^−/− mouse hearts, Gα_i2 mRNA (127±5%) and protein (131±10%) levels were slightly enhanced. Interestingly, L-VDCC current density in cardiomyocytes from Gα_i2 ^−/− mice was lowered (−7.9±0.6 pA/pF, n = 11, p<0.05) compared to wild-type cells (−10.7±0.5 pA/pF, n = 22), whereas it was increased in myocytes from Gα_i3 ^−/− mice (−14.3±0.8 pA/pF, n = 14, p<0.05). Steady-state inactivation was shifted to negative potentials, and recovery kinetics slowed in the absence of Gα_i2 (but not of Gα_i3) and following treatment with pertussis toxin in Gα_i3 ^−/−. The pore forming Ca_vα₁ protein level was unchanged in all mouse models analyzed, similar to mRNA levels of Ca_vα₁ and Ca_vβ₂ subunits. Interestingly, at the cellular signalling level, phosphorylation assays revealed abolished carbachol-triggered activation of ERK1/2 in mice lacking Gα_i2.

Conclusion

Our data provide novel evidence for an isoform-specific modulation of L-VDCC by Gα_i proteins. In particular, loss of Gα_i2 is reflected by alterations in channel kinetics and likely involves an impairment of the ERK1/2 signalling pathway.     

Solid Phase Synthesis and Circular Dichroism Analysis of (i → i + 4) Cyclic Lactam Analogues of Kisspeptin

The α-helix is one of the most common secondary structure elements adopted by proteins and is commonly stabilized in synthetic peptides via the formation of a covalent side-chain to side-chain lactam bridge. In this study, we explored the application of various side-chain to side-chain lactam bridges to helix stabilization of kisspeptin analogues, an interesting candidate for ligand-based drug discovery with potential as anti-metastatic agents. We successfully synthesised a series of Asp/Lys, Lys/Asp, Glu/Lys and Lys/Glu lactams, finding peptide (1) cyclo(4,8)Tyr-Asn-Trp-Glu-Ala-Phe-Gly-Lys-Arg-Phe-NH₂, to exhibit characteristic α-helical activity in aqueous buffer, in comparison to the linear native peptide, which showed no helical character.     

Improved identification of agonist-mediated Gα(i)-specific human G-protein-coupled receptor signaling in yeast cells by flow cytometry

Flow cytometry enables comparative quantification, population analysis, and high-throughput screening of agonist-mediated G-protein-coupled receptor (GPCR) signaling in genetically engineered yeasts. By using flow cytometry, we found that transformation of yeast cells with a low plasmid number is critical both for the construction of large screening libraries and for stable signal transmission in cell ensembles. Based on these findings, we constructed an engineered yeast strain for the improved identification of signal promotion by Gα(i)-specific human GPCRs using flow cytometry.     

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.     

Sensitization of adenylate cyclase: a general mechanism of neuroadaptation to persistent activation of Galpha(i/o)-coupled receptors?

Acute activation of Galphas-coupled receptors stimulates cyclic AMP accumulation leading to the activation of downstream signaling cascades. These Galphas-mediated events can be countered by acute activation of inhibitory G proteins (Galpha(i/o)), which inhibit the activity of adenylate cyclase, thereby attenuating cyclic AMP accumulation. Furthermore, an additional, less direct mechanism for Galpha(i/o) proteins modulation of cyclic AMP signaling also has been described. Persistent activation of several Galpha(i/o)-coupled receptors has been shown to result in a subsequent paradoxical enhancement of adenylate cyclase activity in response to drug-stimulated cyclic AMP accumulation. This sensitization of adenylate cyclase likely represents a cellular adaptive response following prolonged activation of inhibitory receptors. Recent advances in our knowledge of G protein signaling, adenylate cyclase regulation, and other cellular signaling mechanisms have extensively increased our insight into this phenomenon. It is now thought that sensitization occurs as part of a compensatory mechanism by which the cell adapts to chronic inhibitory input. Such a mechanism may be involved in modulating Galphas-coupled receptor signaling following neurotransmitter elevations that occur in psychiatric disease states or following the administration of many drugs of abuse. This review will focus on recent advances in the understanding of molecular signaling pathways that are involved in sensitization and describe the potential role of sensitization in neuronal cell function.     

Effect of 1-Methyladenine on Localization of Gαi Protein in Starfish Oocytes

Localization and quantitative dynamics of _i subunit of G protein was studied by electron immunocytochemistry and immunoenzyme assay after hormonal induction of oocyte maturation in starfish Asterias amurensis. G_i protein was chiefly localized in the plasma membrane of immature oocytes; 1-methyladenine induced redistribution of the _i protein from the plasma membrane to intracellular structure up to the breakdown of the germinal vesicle.     

Influence of Matrix Metalloproteinase-1 Gene −1607 (1G/2G) (rs1799750) Promoter Polymorphism on Circulating Levels of MMP-1 in Chronic Pancreatitis

This study investigated the role of ?1607 (1G/2G) (rs1799750) polymorphism of the MMP-1 gene in chronic pancreatitis. We genotyped 100 patients with chronic pancreatitis and 100 control subjects using tetra-primer ARMS-PCR followed by agarose gel electrophoresis. Serum levels of MMP-1 were determined by Elisa. Statistical analysis was applied to test the significance of the results. The genotypic and allelic distribution varied significantly between the disease group and the control subjects [OD = 1.981 (1.236–3.181), p = 0.004]. MMP-1 levels were higher in subjects homozygous for the 2G allele than in subjects with the 1G allele. The present study revealed a significant association of the MMP-1 ?1607 1G/2G (rs1799750) gene promoter polymorphism with chronic pancreatitis, and it can be considered a biological marker in the etiology of chronic pancreatitis.     

Evidence for a Second, High Affinity G�¦� Binding Site on G��i1(GDP) Subunits

It is well known that Gα_i1(GDP) binds strongly to Gβγ subunits to form the Gα_i1(GDP)-Gβγ heterotrimer, and that activation to Gα_i1(GTP) results in conformational changes that reduces its affinity for Gβγ subunits. Previous studies of G protein subunit interactions have used stoichiometric amounts of the proteins. Here, we have found that Gα_i1(GDP) can bind a second Gβγ subunit with an affinity only 10-fold weaker than the primary site and close to the affinity between activated Gα_i1 and Gβγ subunits. Also, we find that phospholipase Cβ2, an effector of Gβγ, does not compete with the second binding site implying that effectors can be bound to the Gα_i1(GDP)-(Gβγ)₂ complex. Biophysical measurements and molecular docking studies suggest that this second site is distant from the primary one. A synthetic peptide having a sequence identical to the putative second binding site on Gα_i1 competes with binding of the second Gβγ subunit. Injection of this peptide into cultured cells expressing eYFP-Gα_i1(GDP) and eCFP-Gβγ reduces the overall association of the subunits suggesting this site is operative in cells. We propose that this second binding site serves to promote and stabilize G protein subunit interactions in the presence of competing cellular proteins.The plasma membranes of cells are organized as a series of protein-rich and lipid-rich domains (1–3). Many of the protein-rich domains, in particular those organized by caveolin proteins, are thought to be complexes of functionally related proteins that transduce extracellular signals (2). There is increasing evidence that heterotrimeric G proteins exist in pre-formed membrane complexes with their receptors and their intracellular effectors (4–8).The G protein signaling system is initiated when an extracellular agonist binds to its specific G protein-coupled receptor (for review see Refs. 9–12). The ligand-bound receptor will then catalyze the exchange of GTP for GDP on the Gα subunit in the G protein heterotrimer. In the basal state, Gα(GDP) binds strongly to Gβγ, but in the GTP-bound state this affinity is reduced, allowing Gα(GTP) and Gβγ subunits to individually bind to a host of specific intracellular enzymes and change their catalytic activity.Although the interactions between G protein subunits have been studied extensively in vitro, their behavior in cells may differ. For example, in pure or semi-pure systems, activation of Gα(GDP) sufficiently weakens its affinity for Gβγ resulting in dissociation (13). However, in cells separation of the heterotrimer is observed under some circumstances, but not others (7, 14–17). The reason for these differences in behavior is not clear. There are four families of Gα subunits that each contain several members, and, additionally, there are many subtypes of Gβγ subunits (18). It is possible that differences in dissociation behavior reflect differences in affinity between G protein subunit subtypes (19), the presence of various protein partners, and/or differences in post-synthetic modifications of the subunits (20).The mechanism that allows activated G proteins to remain bound is not apparent from the crystal structure (21, 22). If G protein subunits do not dissociate in cells, then their interaction must change in such a manner as to expose the effector interaction site(s). We have found that phospholipase Cβ1 (PLCβ1),⁴ an important effector of Gα_q (23), is bound to Gα_q prior to activation and throughout the activation cycle (6) implying that Gα_q(GDP) interacts with PLCβ1 in a non-functional manner.We have evidence that signaling complexes are stabilized by a series of secondary interactions. Using purified proteins and model membranes, we have found that membranes of the Gα_q-Gβγ/PLCβ1/RGS4 signaling system have secondary, weaker binding sites to members of this signaling system in addition to their high affinity site(s) to their functional partner(s). We speculate that secondary contacts allow for self-scaffolding of signaling proteins. To understand the nature of these secondary contacts, we have studied the ability of the Gα_i1(GDP)-Gβγ heterotrimer to remain complexed through the activation cycle (24). Here, we present evidence that Gα_i1(GDP) has two distinct Gβγ binding sites that only differ in affinity by an order of magnitude and may allow for continued association between the subunits upon activation. We also find that this site plays an important role in stabilizing G protein associations in cells and provides a mechanism of self-scaffolding.