Activation Of Gi Research Articles

Receptor-Mediated Vascular Smooth Muscle Migration Induced by LPA Involves p38 Mitogen-Activated Protein Kinase Pathway Activation

Lysophosphatidic acid (LPA), a naturally occurring glycerophospholipid, can evoke various biological responses, including cell migration, proliferation and survival, via activation of G protein-coupled receptors (GPCRs). However, the role of LPA receptors and details of LPA signaling in migration are largely unexplored. In this study we detect the expression of LPA1 and LPA3 receptors in rat aortic smooth muscle cells (RASMCs). LPA stimulated RASMCs migration in a dose-dependent manner and induced the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and extracellular signal-regulated kinase (ERK). LPA-induced cell migration was significantly inhibited by specific LPA1/LPA3-receptor antagonist Dioctylglycerol pyrophosphate (8:0) (DGPP8.0) at higher concentration. Migration of cells toward LPA was partially, but significantly, reduced in the presence of SB-203580, a p38 MAPK inhibitor, but not PD98059, an ERK inhibitor. In addition, pertussis toxin (PTX), a Gi protein inhibitor, induced an inhibitory effect on p38 MAPK, ERK phosphorylation and RASMCs migration. These data suggest that LPA-induced migration is mediated through the Gi-protein-coupled LPA1 receptor involving activation of a PTX-sensitive Gi / p38MAPK pathway.

Cholinergic receptor signaling modulates spontaneous firing of sinoatrial nodal cells via integrated effects on PKA-dependent Ca2+ cycling and IKACh

Prior studies indicate that cholinergic receptor (ChR) activation is linked to beating rate reduction (BRR) in sinoatrial nodal cells (SANC) via 1) a G(i)-coupled reduction in adenylyl cyclase (AC) activity, leading to a reduction of cAMP or protein kinase A (PKA) modulation of hyperpolarization-activated current (I(f)) or L-type Ca(2+) currents (I(Ca,L)), respectively; and 2) direct G(i)-coupled activation of ACh-activated potassium current (I(KACh)). More recent studies, however, have indicated that Ca(2+) cycling by the sarcoplasmic reticulum within SANC (referred to as a Ca(2+) clock) generates rhythmic, spontaneous local Ca(2+) releases (LCR) that are AC-PKA dependent. LCRs activate Na(+)-Ca(2+) exchange (NCX) current, which ignites the surface membrane ion channels to effect an AP. The purpose of the present study was to determine how ChR signaling initiated by a cholinergic agonist, carbachol (CCh), affects AC, cAMP, and PKA or sarcolemmal ion channels and LCRs and how these effects become integrated to generate the net response to a given intensity of ChR stimulation in single, isolated rabbit SANC. The threshold CCh concentration ([CCh]) for BRR was approximately 10 nM, half maximal inhibition (IC(50)) was achieved at 100 nM, and 1,000 nM stopped spontaneous beating. G(i) inhibition by pertussis toxin blocked all CCh effects on BRR. Using specific ion channel blockers, we established that I(f) blockade did not affect BRR at any [CCh] and that I(KACh) activation, evidenced by hyperpolarization, first became apparent at [CCh] > 30 nM. At IC(50), CCh reduced cAMP and reduced PKA-dependent phospholamban (PLB) phosphorylation by approximately 50%. The dose response of BRR to CCh in the presence of I(KACh) blockade by a specific inhibitor, tertiapin Q, mirrored that of CCh to reduced PLB phosphorylation. At IC(50), CCh caused a time-dependent reduction in the number and size of LCRs and a time dependent increase in LCR period that paralleled coincident BRR. The phosphatase inhibitor calyculin A reversed the effect of IC(50) CCh on SANC LCRs and BRR. Numerical model simulations demonstrated that Ca(2+) cycling is integrated into the cholinergic modulation of BRR via LCR-induced activation of NCX current, providing theoretical support for the experimental findings. Thus ChR stimulation-induced BRR is entirely dependent on G(i) activation and the extent of G(i) coupling to Ca(2+) cycling via PKA signaling or to I(KACh): at low [CCh], I(KACh) activation is not evident and BRR is attributable to a suppression of cAMP-mediated, PKA-dependent Ca(2+) signaling; as [CCh] increases beyond 30 nM, a tight coupling between suppression of PKA-dependent Ca(2+) signaling and I(KACh) activation underlies a more pronounced BRR.

Anastellin, the Angiostatic Fibronectin Peptide, Is a Selective Inhibitor of Lysophospholipid Signaling

Angiogenesis is regulated by integrin-dependent cell adhesion and the activation of specific cell surface receptors on vascular endothelial cells by angiogenic factors. Lysophosphatidic acid (LPA) and sphingosine-1 phosphate (S1P) are bioactive lysophospholipids that activate G protein-coupled receptors that stimulate phosphatidylinositol 3-kinase (PI3K), Ras, and Rho effector pathways involved in vascular cell survival, proliferation, adhesion, and migration. Previous studies have shown that anastellin, a fragment of the first type III module of fibronectin, functions as an antiangiogenic peptide suppressing tumor growth and metastasis. We have previously shown that anastellin blocks serum-dependent proliferation of microvessel endothelial cells (MVEC) by affecting extracellular signal-regulated kinase (ERK)-dependent G(1)-S transition. However, the mechanism by which anastellin regulates endothelial cell function remains unclear. In the present study, we mapped several lysophospholipid-mediated signaling pathways in MVEC and examined the effects of anastellin on LPA- and S1P-induced MVEC proliferation, migration, and cytoskeletal organization. Both LPA and S1P activated PI3K, Ras/ERK, and Rho/Rho kinase pathways, leading to migration, G(1)-S cell cycle progression, and stress fiber formation, respectively. Stimulation of proliferation by LPA/S1P occurred through a G(i)-dependent Ras/ERK pathway, which was independent of growth factor receptors and PI3K and Rho/Rho kinase signaling. Although LPA and S1P activated both PI3K/Akt and Ras/ERK signaling through G(i), anastellin inhibited only the Ras/ERK pathway. Stress fiber formation in response to LPA was dependent on Rho/Rho kinase but independent of G(i) and unaffected by anastellin. These results suggest that lysophospholipid mediators of G(i) activation leading to PI3K/Akt and Ras/ERK signaling bifurcate downstream of G(i) and that anastellin selectively inhibits the Ras/ERK arm of the pathway.

Modeling of G-protein-coupled Receptor Signaling Pathways

Modeling of G-protein-coupled Receptor Signaling Pathways

S1P/S1P 1 signaling stimulates cell migration and invasion in Wilms tumor

Sphingosine-1-phosphate (S1P) is an important regulator of cellular functions via interaction with its receptors S1P 1–5. To date, nothing is known about the S1P receptor expression and the effects of S1P signaling in Wilms tumor. In this study, we found ubiquitous expression of S1P receptors in Wilms tumor specimens and cell lines. We demonstrated that S1P 1 acted as a promigratory modulator by employing S1P 1 antagonist VPC44116, S1P 1 siRNA and adenoviral transduction in Wilms tumor cells. Further, we clarified that S1P 1-mediated migration occurred via Gi coupling and activation of PI3K and Rac1. In addition, S1P stimulated WiT49 cell invasion through S1P 1/Gi signaling pathway. We consider that targeting S1P 1 may be a point of therapeutic intervention in Wilms tumor.

Receptor-Mediated Changes at the Myristoylated Amino Terminus of Gαil Proteins

G protein-coupled receptors (GPCRs) catalyze nucleotide release in heterotrimeric G proteins, the slow step in G protein activation. G i/o family proteins are permanently, cotranslationally myristoylated at the extreme amino terminus. While myristoylation of the amino terminus has long been known to aid in anchoring G i proteins to the membrane, the role of myristoylation with regard to interaction with activated receptors is not known. Previous studies have characterized activation-dependent changes in the amino terminus of Galpha proteins in solution [Medkova, M. (2002) Biochemistry 41, 9963-9972; Preininger, A. M. (2003) Biochemistry 42, 7931-7941], but changes in the environment of specific residues within the Galpha i1 amino terminus during receptor-mediated G i activation have not been reported. Using site-specific fluorescence labeling of individual residues along a stretch of the Galpha il amino terminus, we found specific changes in the environment of these residues upon interaction with the activated receptor and following GTPgammaS binding. These changes map to a distinct surface of the amino-terminal helix opposite the Gbetagamma binding interface. The receptor-dependent fluorescence changes are consistent with a myristoylated amino terminus in the proximity of the membrane and/or receptor. Myristoylation affects both the rate and intensity of receptor activation-dependent changes detected at several residues along the amino terminus (with no significant effect on the rate of receptor-mediated GTPgammaS binding). This work demonstrates that the myristoylated amino terminus of Galpha il proteins undergoes receptor-mediated changes during the dynamic process of G protein signaling.

First Cytoplasmic Loop of Glucagon‐like Peptide‐1 Receptor Can Function at the Third Cytoplasmic Loop Position of Rhodopsin†

G protein-coupled receptors (GPCRs) are classified into several families based on their amino acid sequences. In family 1, GPCRs such as rhodopsin and adrenergic receptor, the structure-function relationship has been extensively investigated to demonstrate that exposure of the third cytoplasmic loop is essential for selective G protein activation. In contrast, much less is known about other families. Here we prepared chimeric mutants between Gt-coupled rhodopsin and Gi/Go- and Gs-coupled glucagon-like peptide-1 (GLP-1) receptor of family 2 and tried to identify the loop region that functions at the third cytoplasmic loop position of rhodopsin. We succeeded in expressing a mutant having the first cytoplasmic loop of GLP-1 receptor and found that this mutant activated Gi and Go efficiently but did not activate Gt. Moreover, the rhodopsin mutant having the first loop of Gs-coupled secretin receptor of family 2 decreased the Gi and Go activation efficiencies. Therefore, the first loop of GLP-1 receptor would share a similar role to the third loop of rhodopsin in G protein activation. This result strongly suggested that different families of GPCRs have maintained molecular architectures of their ancestral types to generate a common mechanism, namely exposure of the cytoplasmic loop, to activate peripheral G protein.

Identification of Farnesyl Pyrophosphate and N-Arachidonylglycine as Endogenous Ligands for GPR92

A series of small compounds acting at the orphan G protein-coupled receptor GPR92 were screened using a signaling pathway-specific reporter assay system. Lipid-derived molecules including farnesyl pyrophosphate (FPP), N-arachidonylglycine (NAG), and lysophosphatidic acid were found to activate GPR92. FPP and lysophosphatidic acid were able to activate both G(q/11)- and G(s)-mediated signaling pathways, whereas NAG activated only the G(q/11)-mediated signaling pathway. Computer-simulated modeling combined with site-directed mutagenesis of GPR92 indicated that Thr(97), Gly(98), Phe(101), and Arg(267) of GPR92 are responsible for the interaction of GPR92 with FPP and NAG. Reverse transcription-PCR analysis revealed that GPR92 mRNA is highly expressed in the dorsal root ganglia (DRG) but faint in other brain regions. Peripheral tissues including, spleen, stomach, small intestine, and kidney also expressed GPR92 mRNA. Immunohistochemical analysis revealed that GPR92 is largely co-localized with TRPV1, a nonspecific cation channel that responds to noxious heat, in mouse and human DRG. FPP and NAG increased intracellular Ca(2+) levels in cultured DRG neurons. These results suggest that FPP and NAG play a role in the sensory nervous system through activation of GPR92.

Characterization of an Orphan G Protein-coupled Receptor, GPR20, That Constitutively Activates Gi Proteins

GPR20 was isolated as an orphan G protein-coupled receptor from genomic DNA by PCR amplification. Although GPR20 was closely related to nucleotide or lipid receptors, the functional role of this receptor, as well as its endogenous ligand, remains unclear. Here we demonstrate that GPR20 is constitutively active in the absence of ligand, leading to continuous activation of its coupled G proteins. When GPR20 was exogenously expressed in HEK293 cells, both the basal level and the prostaglandin E(2)-induced production of cAMP were significantly decreased. A remarkable increase in [(35)S]guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) binding to membrane preparations was also observed in GPR20-expressing cells. These effects of GPR20 overexpression were diminished in cells treated with pertussis toxin, suggesting that the expression of GPR20 results in the activation of G(i/o) proteins. Involvement of GPR20 in the activation of G(i/o) proteins was also supported by evidence that the disruption of a conserved DRY motif in GPR20 attenuated both [(35)S]GTPgammaS incorporation and inhibition of the prostaglandin E(2)-induced cAMP production. Knockdown of GPR20 in PC12h cells resulted in an elevation of the basal cAMP level, suggesting that the endogenous GPR20 achieves a constitutively or spontaneously active conformation. Furthermore, enhancement of [(3)H]thymidine incorporation was also observed in the GPR20-silencing cells, implying that the GPR20 expression seems to attenuate PC12h cell growth. Taken together, these data indicate that GPR20 constitutively activates G(i) proteins without ligand stimulation. The receptor may be involved in cellular processes, including control of intracellular cAMP levels and mitogenic signaling.

Targeted Inhibition of Cardiomyocyte Gi Signaling Enhances Susceptibility to Apoptotic Cell Death in Response to Ischemic Stress

A salient characteristic of dysfunctional myocardium progressing to heart failure is an upregulation of the adenylyl cyclase inhibitory guanine nucleotide (G) protein alpha subunit, G alpha(i2). It has not been determined conclusively whether increased Gi activity in the heart is beneficial or deleterious in vivo. Gi signaling has been implicated in the mechanism of cardioprotective agents; however, no in vivo evidence exists that any of the G alpha subunits are cardioprotective. We have created a novel molecular tool to specifically address the role of Gi proteins in normal and dysfunctional myocardium. We have developed a class-specific Gi inhibitor peptide, GiCT, composed of the region of G alpha(i2) that interacts specifically with G protein-coupled receptors. GiCT inhibits Gi signals specifically in vitro and in vivo, whereas Gs and Gq signals are not affected. In vivo expression of GiCT in transgenic mice effectively causes a "functional knockout" of cardiac G alpha(i2) signaling. Inducible, cardiac-specific GiCT transgenic mice display a baseline phenotype consistent with nontransgenic mice. However, when subjected to ischemia/reperfusion injury, GiCT transgenic mice demonstrate a significant increase in infarct size compared with nontransgenic mice (from 36.9+/-2.5% to 50.9+/-4.3%). Mechanistically, this post-ischemia/reperfusion phenotype includes increased myocardial apoptosis and resultant decreased contractile performance. Overall, our results demonstrate the in vivo utility of GiCT to dissect specific mechanisms attributed to Gi signaling in stressed myocardium. Our results with GiCT indicate that upregulation of G alpha(i2) is an adaptive protective response after ischemia to shield myocytes from apoptosis.

Lysophosphatidic acid induced oxidized low‐density lipoprotein uptake is mediated through activation of Gi and expression of scavenger receptor class‐A in mouse macrophages

Lysophosphatidic acid (LPA) is a platelet‐, and oxidized low‐density lipoprotein (ox‐LDL)‐enriched lysophospholipid (LPL). By binding to its cognate G protein coupled‐receptors, LPA is known to regulate multiple biological functions, including inflammation, and atherosclerosis. Atherosclerosis is an inflammatory disease, and aberrantly uptake of ox‐LDL by macrophages is crucial in this process. Nevertheless, how LPA regulates macrophages in the progress of atherosclerosis is currently unknown. In this study, we found that LPA induced DiI fluorescence‐labeled ox‐LDL uptake in mouse J774A. 1 macrophages through activation of Gi‐coupled LPA receptors by using flow cytometry analysis. Meanwhile, both mRNA and protein levels of scavenger receptor class‐A (SR‐A), a receptor for modified LDL in macrophages, were also enhanced by LPA treatment. In addition, the enhancement effects of ox‐LDL uptake by LPA were significantly inhibited by SR‐A functional blocking antibody. In conclusion, our results suggested that LPA is a pro‐atherogenic factor through activating SR‐A expression in macrophages.

A Luminescent cAMP Assay for determining cellular and tissue cAMP and monitoring the Modulation of Gs and Gi Protein Coupled Receptors

G Protein Coupled Receptors represent a large validated target for Drug discovery research. The Gs is coupled to activation of adenylate cyclase and Gi is coupled to inhibition of adenylate cyclase while Gq is coupled to activation of phospholipase ƒÒ. Cellular concentrations of second messengers such as cAMP and cGMP have been known to play major roles in smooth muscle relaxation, contractile activity of cardiovascular system, and central nervous system related pathologies. We report here on a new assay for determining cAMP concentrations in cellular and tissue extract and for monitoring modulation of GPCR that are linked to activation or inhibition of adenylate cyclase (Gs or Gi). The assay is homogenous, fast, HTS formatted with high Z¡¦, and adaptable to multi plate formats. We have successfully generated EC50 values for agonists and IC50 values for antagonists of Gs coupled receptors that are similar to those reported in the literature. The assay is easy to use, and can be carried out in less than 60 minutes and does not require antibodies or expensive instrumentation for signal detection and the signal output is relatively stable for few hours.

Characterization of the vasoactive properties of S1P receptors in rat aorta

In the cardiovascular system, the bioactive lipid sphingosine‐1‐phosphate (S1P) exerts its vasoactive effects predominantly via the S1P1, S1P2 and S1P3 receptor which are differentially expressed in vascular smooth muscle cells (VSMCs) and endothelial cells (ECs).We have explored the contribution of these three receptors to vascular reactivity in rat aorta and to related signaling mechanisms in isolated VSMCs and ECs using some new S1P receptor ligands. In rat aortic rings, all agonists at the S1P1 and/or S1P3 receptor induced endothelium‐dependent vasorelaxation in pre‐contracted preparations. In rat aortic ECs, which showed a high mRNA expression of the Gi‐coupled S1P1 receptor, all ligands decreased the forskolin‐induced cAMP accumulation whereas no major changes in [Ca2+]i were observed. In VSMCs, which express mRNA for all three S1P receptors, predominantly Gq‐coupled S1P2 receptor signaling was observed, resulting in increases in [Ca2+]i.In conclusion, the compounds tested in this study can act on both ECs and VSMCs from rat aorta. S1P1 receptor stimulation in ECs leads to Gi activation probably resulting in relaxation via activation of PI3K/Akt and subsequent NO production. Stimulation of mainly the S1P2 receptor in VSMCs results in calcium elevation, a response typically linked to contraction. For agonists devoid of S1P2 activity, the net effect observed on aortic tone is thus vasorelaxation.

Both Gi and Go Heterotrimeric G Proteins Are Required to Exert the Full Effect of Norepinephrine on the β-Cell KATP Channel

The effects of norepinephrine (NE), an inhibitor of insulin secretion, were examined on membrane potential and the ATP-sensitive K+ channel (K ATP) in INS 832/13 cells. Membrane potential was monitored under the whole cell current clamp mode. NE hyperpolarized the cell membrane, an effect that was abolished by tolbutamide. The effect of NE on K ATP channels was investigated in parallel using outside-out single channel recording. This revealed that NE enhanced the open activities of the K ATP channels approximately 2-fold without changing the single channel conductance, demonstrating that NE-induced hyperpolarization was mediated by activation of the K ATP channels. The NE effect was abolished in cells preincubated with pertussis toxin, indicating coupling to heterotrimeric G i/G o proteins. To identify the G proteins involved, antisera raised against alpha and beta subunits (anti-G alpha common, anti-G beta, anti-G alpha i1/2/3, and anti-G alpha o) were used. Anti-G alpha common totally blocked the effects of NE on membrane potential and K ATP channels. Individually, anti-G alpha i1/2/3 and anti-G alpha o only partially inhibited the action of NE on K ATP channels. However, the combination of both completely eliminated the action. Antibodies against G beta had no effects. To confirm these results and to further identify the G protein subunits involved, the blocking effects of peptides containing the sequence of 11 amino acids at the C termini of the alpha subunits were used. The data obtained were similar to those derived from the antibody work with the additional information that G alpha i3 and G alpha o1 were not involved. In conclusion, both G i and G o proteins are required for the full effect of norepinephrine to activate the K ATP channel.

Dynamic Regulation, Desensitization, and Cross-talk in Discrete Subcellular Microdomains during β2-Adrenoceptor and Prostanoid Receptor cAMP Signaling

Dynamic and localized actions of cAMP are central to the generation of discrete cellular events in response to a range of G(s)-coupled receptor agonists. In the present study we have employed a cyclic nucleotide-gated channel sensor to report acute changes in cAMP in the restricted cellular microdomains adjacent to two different G(s)-coupled receptor pathways, beta(2)-adrenoceptors and prostanoid receptors that are expressed endogenously in HEK293 cells. We probed by either selective small interference RNA-mediated knockdown or dominant negative overexpression the contribution of key signaling components in the rapid attenuation of the local cAMP signaling and subsequent desensitization of each of these G-protein-coupled receptor signaling pathways immediately following receptor activation. Direct measurements of cAMP changes just beneath the plasma membrane of single HEK293 cells reveal novel insights into key regulatory roles provided by protein kinase A-RII, beta-arrestin2, cAMP phosphodiesterase-4D3, and cAMP phosphodiesterase-4D5. We provide new evidence for distinct modes of cAMP down-regulation in these two G(s)-linked pathways and show that these distinct G-protein-coupled receptor signaling systems are subject to unidirectional, heterologous desensitization that allows for limited cross-talk between distinct, dynamically regulated pools of cAMP.

ROLES OF α1A‐ AND α1B‐ADRENOCEPTORS IN HEART: INSIGHTS FROM STUDIES OF GENETICALLY MODIFIED MICE

1. Several mouse strains have been prepared in which different subtypes of the alpha1-adrenoceptor (AR) are overexpressed or deleted. The phenotypes of the animals generated vary depending on whether the receptors are expressed specifically in heart or generally throughout the animal, but some overall conclusions can be drawn. 2. Heightened activity of alpha1B-AR by overexpressing the receptors leads to depressed contractile responses to beta-AR activation, which may be related to activation of the inhibitory G-protein Gi. In contrast, alpha1A-AR cause substantially heightened contractility when overexpressed in heart. 3. Overexpressed alpha1B-AR predispose hearts to hypertrophy and worsen heart failure caused by pressure overload, whereas increased alpha1A-AR expression does not influence hypertrophic responses and, furthermore, improves outcomes after pressure overload or myocardial infarction. 4. Alpha1A-adrenoceptors mediate a preconditioning action to improve functional recovery after acute ischaemic insult, whereas alpha1B-AR are ineffective. Both subtypes appear to protect from inositol 1,4,5-trisphosphate generation and arrhythmogenesis in early postischaemic reperfusion. 5. Although some of the protective effects of heightened alpha1A-AR drive may be related to the enhanced contractility, it is also possible that alpha1A-AR protect from cardiomyocyte apoptotic responses.

Achieving signalling selectivity of ligands for the corticotropin‐releasing factor type 1 receptor by modifying the agonist's signalling domain

Most of the pharmaceuticals target G-protein-coupled receptors (GPCRs) which can generally activate different signalling events. The aim of this study was to achieve functional selectivity of corticotropin-releasing factor receptor type 1 (CRF(1)) ligands. We systematically substituted urocortin, a natural peptide agonist of CRF(1), with bulky amino acids (benzoyl-phenylalanine, naphthylalanine) and determined the effect of the analogues on coupling of CRF(1) to Gs- and Gi-protein in human embryonic kidney cells, using receptor binding, [(35)S]-GTPgammaS binding stimulation, and cAMP accumulation assays. Native ligands stimulated Gs and Gi activation through CRF(1), resulting in stimulation and then inhibition of cAMP accumulation. Single replacements in urocortin at positions 6-15 led, dependent on the position and nature of the substituent, to ligands that conserved Gs activity, but were devoid of Gi activity, only stimulating cAMP accumulation, and competitively antagonized the Gi activation by sauvagine. In contrast, analogues with substitutions outside this sequence non-selectively activated Gs and Gi, as urocortin did. Modifications in a specific region, which we have called the signalling domain, in the polypeptide agonist urocortin resulted in analogues that behaved as agonists and, at the same time, antagonists for the activation of different G-proteins by CRF(1). This finding implies significant differences between active conformations of the receptor when coupled to different G-proteins. A similar structural encoding of signalling information in other polypeptide hormone receptor ligands would result in a general concept for the development of signalling-selective drug candidates.

Oligomerization of neuropeptide Y (NPY) Y2 receptors in CHO cells depends on functional pertussis toxin-sensitive G-proteins

Human neuropeptide Y Y2 receptors expressed in CHO cells are largely oligomeric, and upon solubilization are recovered by density gradient centrifugation as ∼ 180 kDa complexes of receptor dimers and G-protein heterotrimers. A large fraction of the receptors is inactivated in the presence of pertussis toxin, in parallel with inactivation of Gi α subunits (with half-periods of about 4 h for both). This is accompanied by a very long-lasting loss of receptor dimers and of masked surface Y2 sites (an apparent receptor reserve pre-coupled mainly to Gi α subunit-containing G-proteins). However, surface Y2 receptors accessible to large peptide agonists are much less sensitive to the toxin. All surface Y2 receptors are rapidly blocked by Y2 antagonist BIIE0246, with a significant loss of the dimers, but with little change of basal Gi activity. However, both dimers and Y2 receptor compartmentalization are restored within 24 h after removal of the antagonist. In CHO cells, the maintenance and organization of Y2 receptors appear to critically depend on functional pertussis toxin-sensitive G-proteins.

Identification of the Intracellular Region of the Leukotriene B4 Receptor Type 1 That Is Specifically Involved in Gi Activation

Many G-protein-coupled receptors can activate more than one G-protein subfamily member. Leukotriene B4 receptor type 1 (BLT1) is a high affinity G-protein-coupled receptors for leukotriene B4 functioning in host defense, inflammation, and immunity. Previous studies have shown that BLT1 utilizes different G-proteins (the Gi family and G16 G-proteins) in mediating diverse cellular events and that truncation of the cytoplasmic tail of BLT1 does not impair activation of Gi and G16 proteins. To determine responsive regions of BLT1 for G-protein coupling, we performed an extensive mutagenesis study of its intracellular loops. Three intracellular loops (i1, i2, and i3) of BLT1 were found to be important for both Gi and G16 coupling, as judged by Gi-dependent guanosine 5'-(gamma-thio) triphosphate (GTPgammaS) binding and G16-dependent inositol phosphate accumulation assays. The i3-1 mutant, with a mutation at the i3 amino terminus, exhibited greatly reduced GTPgammaS binding but intact inositol phosphate accumulation triggered by leukotriene B4 stimulation. These results suggest that the i3-1 region is required only for Gi activation. Moreover, in the i3-1 mutant, the deficiency in Gi activation was accompanied by a loss of the high affinity leukotriene B4 binding state seen with the wild type receptor. A three-dimensional model of BLT1 constructed based on the structure of bovine rhodopsin suggests that the i3-1 region may consist of the cytoplasmic end of the transmembrane helix V, which protrudes the helix into the cytoplasm. From mutational studies and three-dimensional modeling, we propose that the extended cytoplasmic helix connected to the transmembrane helix V of BLT1 might be a key region for selective activation of Gi proteins.

Stimulation of extracellular signal-regulated kinases and proliferation in the human gastric cancer cells KATO-III by obestatin

Obestatin, the ghrelin-associated peptide, activates cell proliferation in the gastric cancer cell line KATO-III. The results showed that this peptide induced cell proliferation by mitogen-activated kinase kinase/extracellular signal-regulated kinases1/2 (ERK1/2) phosphorylation. A sequential analysis of the obestatin transmembrane signalling pathway indicated that the ERK1/2 activity is partially blocked after preincubation of the cells with pertussis toxin, as well as by wortmannin (an inhibitor of phosphoinositide 3-kinase (PI3K)), staurosporine (an inhibitor of protein kinase C (PKC)) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2, which inhibits the non receptor tyrosine kinase Src). Upon administration of obestatin, the intracellular levels of phospho-PKCε- and θ-isoenzymes rise with similar time-courses, from which PKCε appears to be the responsible for ERK1/2 response. Based on the experimental data, a signalling pathway involving the consecutive activation of Gi, PI3K, novel PKCε and Src for ERK1/2 activation is proposed. These results point to a functionally active peptide that regulates proliferation of the gastric cancer cells KATO-III.