A GαsCarboxyl-Terminal Peptide Prevents GsActivation by the A2AAdenosine Receptor

Mazzoni, Maria Rosa; Taddei, Simone; Giusti, Laura; Rovero, Paolo; Galoppini, C.; D’Ursi, Anna Maria; Albrizio, Stefania; Triolo, Antonio; Novellino, Ettore; Greco, Giovanni; Lucacchini, Antonio; Hamm, Heidi E.

doi:10.1124/mol.58.1.226

Cited by 40 publications

(45 citation statements)

References 36 publications

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“…Peptides corresponding to the carboxyl terminus of the G␣ subunits of G proteins, which represent an important site of interaction with the receptor, have been reported to specifically uncouple receptors from G proteins in several systems, such as adenosinergic, adrenergic, and serotonergic, leading either to a low-or high-affinity state of the receptors (Gilchrist et al, 1998;Chang et al, 2000;Mazzoni et al, 2000). The G␣ i1,2 G␣ i3 , G␣ o , and G␣ s inhibitory peptides were therefore tested for their ability to modulate the specific binding of 3 H]DAMGO (Fig.…”

Section: Methodsmentioning

confidence: 99%

Neuropeptide FF (NPFF) Analogs Functionally Antagonize Opioid Activities in NPFF₂ Receptor-Transfected SH-SY5Y Neuroblastoma Cells

et al. 2004

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To elucidate the mechanism of the cellular antiopioid activity of neuropeptide FF (NPFF), we have transfected the SH-SY5Y neuroblastoma cell line, which expresses -and ␦-opioid receptors, with the human NPFF 2 receptor. The selected clone, SH 2 -D9, expressed high-affinity NPFF 2 receptors in the same range order as -and ␦-opioid receptors (100 -300 fmol/mg of protein channels by opioid agonists. In the presence of carbachol, acting on muscarinic receptors to release Ca 2ϩ from the intracellular stores, deltorphin-I and 1DMe enhanced this release. Preincubation with 1DMe reduced the maximal effect of deltorphin-I by 40%, demonstrating an antiopioid effect in this experimental model for the first time. By using peptides corresponding to the carboxyl terminus of the ␣ i1,2 , ␣ i3 , ␣ o , and ␣ s subunits of G proteins, which specifically uncouple receptors from G proteins, we demonstrated that -opioid and NPFF 2 receptors couple to the four subunits assayed. The Ca 2ϩ release from the intracellular stores by 1DMe resulted from the coupling of NPFF 2 receptors with G␣ o and G␣ i1,2 , whereas the coupling with G␣ s reduced the antiopioid effect of 1DMe in the modulation of N-type channels. This SH 2 -D9 cell line now provides the opportunity to study the interaction between both receptors.Neuropeptide FF (NPFF), FLFQPQRFamide, is representative of a family of mammalian amidated neuropeptides whose precursors pro-NPFF A and pro-NPFF B and G proteincoupled receptors NPFF 1 and NPFF 2 have been recently cloned (Zajac, 2001). Although NPFF does not interact with opioid receptors (Gouardères et al., 1998), a close relationship between neuropeptide FF and opioid systems has been clearly demonstrated in the central nervous system, especially in pain perception (Harrison et al., 1998;Roumy and Zajac, 1998;Panula et al., 1999). For instance, supraspinal injection of NPFF analogs, which has little or no effect in pain tests, decreases morphine-induced analgesia (antiopioid activity, Roumy and Zajac, 1998;Panula et al., 1999), whereas spinal administration induces a naloxone-sensitive analgesia and potentiates morphine-induced analgesia (proopioid activity, Roumy and Zajac, 1998;Panula et al., 1999).In neurons, opioids, including nociceptin, 1) inhibit adenylyl cyclase activity and 2) stimulate inwardly rectifying K ϩ channels and inhibit voltage-dependent Ca 2ϩ currents by activating four types, , ␦, , and Opioid Receptor-Like 1, of opioid receptors (Law et al., 2000). This leads to postsynaptic neuronal inhibition and to presynaptic inhibition of transmitter release, respectively. In contrast to opioids, no data report a direct modulation of K ϩ and Ca 2ϩ conductances by NPFF or analogs but rather describe a blockade of the opioid activity on ionic conductances when cells are pretreated with NPFF. NPFF or analogs, which are inactive by themselves, reverse the opioid-induced inhibition of Ca 2ϩ conductance in NPFF 2 receptor-expressing neurons dissociated from rat dorThis work was supported by the Centre National de l...

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Section: Methodsmentioning

confidence: 99%

Neuropeptide FF (NPFF) Analogs Functionally Antagonize Opioid Activities in NPFF₂ Receptor-Transfected SH-SY5Y Neuroblastoma Cells

et al. 2004

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“…In isolated plasma membranes, 11-amino acid peptides representing the carboxyl termini of G␣ i1/2 or G␣ o modulate ligand binding to the adenosine A 1 receptor by disrupting the high affinity receptor-G protein complex (16). Similarly, modified 16 -21-amino acid peptides derived from the carboxyl terminus of G␣ s inhibit high affinity agonist binding to the adenosine A 2A receptors, and impair A 2A receptor-mediated adenylyl cyclase activation (17). These data suggest that the isolated carboxyl-terminal ␣-helix can interact with a receptor in a manner that precludes productive receptor-G protein coupling.…”

Section: Discussionmentioning

confidence: 99%

Selective Inhibition of Heterotrimeric GsSignaling

Feldman

Zamah²,

Pierce³

et al. 2002

Journal of Biological Chemistry

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The blockade of heptahelical receptor coupling to heterotrimeric G proteins by the expression of peptides derived from G protein G␣ subunits represents a novel means of simultaneously inhibiting signals arising from multiple receptors that share a common G protein pool. Here we examined the mechanism of action and functional consequences of expression of an 83-amino acid polypeptide derived from the carboxyl terminus of G␣ s (GsCT). In membranes prepared from GsCT-expressing cells, the peptide blocked high affinity agonist binding to ␤ 2 adrenergic receptors (AR) and inhibited ␤ 2 AR-induced [35 S]GTP␥S loading of G␣ s . GsCT expression inhibited ␤ 2 AR-and dopamine D 1A receptor-mediated cAMP production, without affecting the cellular response to cholera toxin or forskolin, indicating that the peptide inhibited receptor-G s coupling without impairing G protein or adenylyl cyclase function. [35 S]GTP␥S loading of G␣ q/11 by ␣ 1B ARs and G␣ i by ␣ 2A ARs and G q/11 -or G i -mediated phosphatidylinositol hydrolysis was unaffected, indicating that the inhibitory effects of GsCT were selective for G s . We next employed the GsCT construct to examine the complex role of G s in regulation of the ERK mitogen-activated protein kinase cascade, where activation of the cAMP-dependent protein kinase (PKA) pathway reportedly produces both stimulatory and inhibitory effects on heptahelical receptor-mediated ERK activation. For the ␤ 2 AR in HEK-293 cells, where PKA activity is required for ERK activation, expression of GsCT caused a net inhibition of ERK activation. In contrast, ␣ 2A AR-mediated ERK activation in COS-7 cells was enhanced by GsCT expression, consistent with the relief of a downstream inhibitory effect of PKA. ERK activation by the G q/11 -coupled ␣ 1B AR was unaffected by GsCT. These findings suggest that peptide G protein inhibitors can provide insights into the complex interplay between G protein pools in cellular regulation.Heptahelical, or G protein-coupled, receptors represent the single most diverse class of cell surface receptors, both evolutionarily and within the human genome. The basic unit of G protein-coupled receptor signaling is composed of three parts as follows: a heptahelical receptor, a heterotrimeric G protein, 1 and an effector, such as a G protein-regulated enzyme or ion channel. The binding of an extracellular agonist ligand to the receptor changes its conformation so as to permit productive coupling with the G protein, thereby catalyzing the exchange of GTP for GDP on the G␣ subunit, and dissociation of G␣-GTP from G␤␥ subunits. Regulation of effectors is achieved through their interaction with free GTP-bound G␣ or G␤␥ subunits. Based upon data from crystallographic, biochemical, and mutagenesis studies, physical coupling of receptor and G protein is thought to involve primarily the second and third intracellular domains of the receptor, which make physical contact with the carboxyl terminus of the G␣ subunit (1-6). In particular, the last ϳ50 amino acids of the G␣ subunit are important ...

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“…Such somatic mutations have been identified in sporadic endocrine tumors (pituitary, thyroid, adrenal), fibrous dysplasia of bone, and the McCune-Albright syndrome (Lyons et al, 1990;Weinstein, 2002. The amino terminus and switch 2 regions interact directly with β-subunit to form the heterotrimer (Lambright et al, 1996;Wall et al, 1995), while the carboxyl terminus is important for receptor interactions (Conklin et al, 1996;Grishina & Berlot, 2000;Mazzoni et al, 2000;Schwindinger et al, 1994;Simonds et al, 1989;Sullivan et al, 1987). cAMP, the major second messenger resulting from G s α signaling, mediates its effects through direct binding and stimulation of several molecules, including protein kinase A (PKA), cAMPregulated guanine nucleotide exchange factors (cAMP-GEFs) (de Rooij et al, 1998;Kawasaki et al, 1998), and ion channels (Sudlow et al, 1993;Wainger et al, 2001).…”

Section: G S αmentioning

confidence: 99%

Studies of the regulation and function of the Gsα gene Gnas using gene targeting technology

Weinstein

Xie

Zhang

et al. 2007

Pharmacology & Therapeutics

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The heterotrimeric G protein α-subunit G s α is ubiquitously expressed and mediates receptorstimulated intracellular cAMP generation. Its gene Gnas is a complex imprinted gene which uses alternative promoters and first exons to generate other gene products, including the G s α isoform XLαs and the chromogranin-like protein NESP55, which are specifically expressed from the paternal and maternal alleles, respectively. G s α itself is imprinted in a tissue-specific manner, being biallelically expressed in most tissues but paternally silenced in a few tissues. Gene targeting of specific Gnas transcripts demonstrates that heterozygous mutation of G s α on the maternal (but not the paternal) allele leads to early lethality, perinatal subcutaneous edema, severe obesity, and multihormone resistance, while the paternal mutation leads to only mild obesity and insulin resistance. These parent-of-origin differences are the consequence of tissue-specific G s α imprinting. XLαs deficiency leads to a perinatal suckling defect and a lean phenotype with increased insulin sensitivity. The opposite metabolic effects of G s α and XLαs deficiency are associated with decreased and increased sympathetic nervous system activity, respectively. NESP55 deficiency has no metabolic consequences. Other gene targeting experiments have shown Gnas to have two independent imprinting domains controlled by two different imprinting control regions. Tissuespecific G s α knockout models have identified important roles for G s α signaling pathways in skeletal development, renal function, and glucose and lipid metabolism. Our present knowledge gleaned from various Gnas gene targeting models are discussed in relation to the pathogenesis of human disorders with mutation or abnormal imprinting of the human ortholog GNAS.

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A Gα_sCarboxyl-Terminal Peptide Prevents G_sActivation by the A_2AAdenosine Receptor

Cited by 40 publications

References 36 publications

Neuropeptide FF (NPFF) Analogs Functionally Antagonize Opioid Activities in NPFF₂ Receptor-Transfected SH-SY5Y Neuroblastoma Cells

Neuropeptide FF (NPFF) Analogs Functionally Antagonize Opioid Activities in NPFF₂ Receptor-Transfected SH-SY5Y Neuroblastoma Cells

Selective Inhibition of Heterotrimeric GsSignaling

Studies of the regulation and function of the Gsα gene Gnas using gene targeting technology

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A GαsCarboxyl-Terminal Peptide Prevents GsActivation by the A2AAdenosine Receptor

Cited by 40 publications

References 36 publications

Neuropeptide FF (NPFF) Analogs Functionally Antagonize Opioid Activities in NPFF2 Receptor-Transfected SH-SY5Y Neuroblastoma Cells

Neuropeptide FF (NPFF) Analogs Functionally Antagonize Opioid Activities in NPFF2 Receptor-Transfected SH-SY5Y Neuroblastoma Cells

Selective Inhibition of Heterotrimeric GsSignaling

Studies of the regulation and function of the Gsα gene Gnas using gene targeting technology

Contact Info

Product

Resources

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A Gα_sCarboxyl-Terminal Peptide Prevents G_sActivation by the A_2AAdenosine Receptor

Neuropeptide FF (NPFF) Analogs Functionally Antagonize Opioid Activities in NPFF₂ Receptor-Transfected SH-SY5Y Neuroblastoma Cells

Neuropeptide FF (NPFF) Analogs Functionally Antagonize Opioid Activities in NPFF₂ Receptor-Transfected SH-SY5Y Neuroblastoma Cells