Cloning, expression and pharmacological characterisation of the mouse somatostatin sst5 receptor

Feuerbach, Dominik; Fehlmann, Dominique; Nunn, Caroline; Siehler, Sandra; Langenegger, Daniel; Bouhelal, Rochdi; Seuwen, Klaus; Hoyer, Daniel

doi:10.1016/s0028-3908(00)00063-0

Cited by 36 publications

(18 citation statements)

References 21 publications

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“…TCF phosphorylation via MAP kinases is necessary for it to bind SRF, and depending on the cell type and growth factor involved, all three families of MAP kinases (extracellular signal-regulated kinases, stress-activated protein kinases and p38) have been shown to be able to activate the TCF via different pathways (Treisman, 1994;Gille et al, 1995;Price et al, 1995;Whitmarsh et al, 1995;1997). In the present study and in previous studies in our laboratory, we have shown that determination of SRIF-induced luciferase expression produces a sensitive marker of agonist efficacy that differentiates between full and partial agonists (Feuerbach et al, 2000;Nunn et al, 2002;2003a-c). SRIF has previously been shown to be able to increase intracellular Ca 2 þ via sst 2 receptors in recombinant cell lines including COS-7 Tomura et al, 1994), F 4 C 1 pituitary cells (Chen et al, 1997), HEK-293 (Chen & Tashjian, 1994), and to a very small extent in CCL39 Chinese hamster lung fibroblasts (Siehler & Hoyer, 1999c).…”

Section: Nunn Et Alsupporting

confidence: 64%

“…The SRE is regulated by transcription factors that are constitutively bound to DNA and are rapidly phosphorylated and activated in response to many extracellular signals including ligands which act at G protein-coupled receptors (Hill & Treisman, 1995;Treisman, 1995;Chai & Tarnawski, 2002). SRIF receptors have been shown in our lab to concentration-dependently increase luciferase expression via the SRE, providing a sensitive marker of agonist efficacy that differentiates between full and partial agonists, and antagonists (Feuerbach et al, 2000;Nunn et al, 2002;2003a-c).…”

Section: Introductionmentioning

confidence: 96%

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Comparison of functional profiles at human recombinant somatostatin sst₂ receptor: simultaneous determination of intracellular Ca²⁺ and luciferase expression in CHO‐K1 cells

Nunn

Cervia

Langenegger

et al. 2004

British J Pharmacology

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1 Somatostatin (somatotropin release inhibiting factor; SRIF) acts via five G protein-coupled receptors (sst 1 -sst 5 ) that modulate multiple cellular effectors. The aim of this study was to compare two functional effects of the human sst 2 receptor stably expressed in CHO-K1 cells in a single experiment using a duplex assay for intracellular calcium and serum response element (SRE)-driven luciferase expression. 2 Intracellular calcium was measured using a fluorometric imaging plate reader II (FLIPR II). SRIF-14 rapidly and transiently increased intracellular calcium with a pEC 50 of 8.7470.03 (n ¼ 52). At 5 h after FLIPR II measurements, luciferase expression was determined. SRIF-14 concentrationdependently increased luciferase expression (pEC 50 ¼ 9.0670.03, n ¼ 52). 3 Natural and synthetic agonist/antagonist ligands for SRIF receptors were tested in the duplex assay. Correlation of agonist potencies and efficacies between the two responses were significant (r 2 ¼ 0.83 and 0.90, pEC 50 and E max , respectively). 4 Pertussis toxin pretreatment reduced SRIF-14/octreotide-mediated intracellular calcium increases by 45-47% and luciferase expression by 95-98%. 5 Thapsigargin pretreatment abolished the SRIF-14/octreotide-mediated intracellular calcium increase but had no effect on luciferase expression. 6 In conclusion, SRIF stimulates an increase in intracellular calcium and SRE-luciferase expression via human sst 2 receptors in CHO-K1 cells.

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Section: Nunn Et Alsupporting

confidence: 64%

Section: Introductionmentioning

confidence: 96%

Comparison of functional profiles at human recombinant somatostatin sst₂ receptor: simultaneous determination of intracellular Ca²⁺ and luciferase expression in CHO‐K1 cells

Nunn

Cervia

Langenegger

et al. 2004

British J Pharmacology

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“…After an overnight incubation, cells were transiently transfected with cDNAs encoding each of the following (unless otherwise noted): R1, R2, G q5i , and an SRE 5x luciferase reporter gene construct. As reported previously, G q5i enables G i/o -coupled receptor activity to be detected using an SRE-luciferase reporter gene (Feuerbach et al, 2000;Hearn et al, 2002). Transfections were done with Lipofectamine reagent (Invitrogen) according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

Point Mutations in Either Subunit of the GABA_BReceptor Confer Constitutive Activity to the Heterodimer

Mukherjee

McBride²,

Beinborn³

et al. 2006

Mol Pharmacol

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The GABA receptor (GABA B R) is a class C G protein-coupled receptor (GPCR) that functions as an obligate heterodimer, composed of two heptahelical subunits, GABA B R subunit 1 (R1) and GABA B R subunit 2 (R2). In this study, we generated and pharmacologically characterized constitutively active GABA B R mutants as novel tools to explore the molecular mechanisms underlying receptor function. A single amino acid substitution, T290K, in the R1 agonist binding domain results in ligand-independent signaling when this mutant subunit is coexpressed with wild-type R2. Introduction of a Y690V mutation in the putative G protein-coupling domain of R2 is sufficient to confer moderate constitutive activity when this subunit is expressed alone. Activity of the Y690V mutant can be markedly enhanced with coexpression of wild-type R1. Coexpression of both mutant subunits (R1-T290K and R2-Y690K) leads to a further increase in basal signaling. Potencies of the full agonists R-(ϩ)-␤-(aminomethyl)-4-chlorobenzenepropanoic acid hydrochloride (baclofen) and GABA are increased at the constitutively active versus the corresponding wild-type receptors. The mutant GABA B R variants provided a sensitive probe enabling detection of inverse or partial agonist activity of molecules previously considered neutral antagonists. Our studies using constitutively active isoforms provide independent support for a model of GABA B R function that takes into account 1) ligand binding by R1, 2) signal transduction by R2, and 3) modulation of R2-induced function by R1. Furthermore, we demonstrate that certain hallmark features of constitutive activity as originally established with class A GPCRs (e.g., enhanced agonist potency and affinity), are more generally applicable, as suggested by our finding with a class C heterodimeric receptor.

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“…Alternatively, cells were transfected with cDNA encoding a wild type or mutant hD2R in combination with Gq5i and an SRE 5x luciferase reporter gene construct (five tandem repeats of the serum response element ligated upstream from a firefly luciferase reporter gene; Feuerbach et al 2000). As reported previously, G q5i enables G i/o -coupled receptor activity to be detected using a serum response element (SRE)-luciferase reporter gene (Conklin et al 1993;Hearn et al 2002;AlFulaij et al 2007).…”

Section: Generation Of Mutant Receptorsmentioning

confidence: 98%

Pharmacological Analysis of Human D1 and D2 Dopamine Receptor Missense Variants

et al. 2008

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Drugs targeting dopamine receptors have been the focus of much research over the past 30 years, in large part because of their role in treating multiple pathological conditions including Parkinson's disease, schizophrenia, Tourette's syndrome, and hyperprolactinemia. Missense mutations in G protein-coupled receptors (GPCRs) can alter basal and/or ligand-induced signaling, which in turn can affect individuals' susceptibility to disease and/or response to therapeutics. To date, five coding variants in the human D1 receptor (hD1R; T37P, T37R, R50S, S199A, and A229T) and three in the human D2 receptor (hD2R; P310S, S311C, and T351A) have been reported in the NCBI single nucleotide polymorphism database. We utilized site-directed mutagenesis to generate cDNAs encoding these receptor isoforms. After expression in either HEK293 or neuronal GT1 cells, basal and ligand-induced signaling of each of these receptors was determined and compared to wild type. In addition, we investigated expression levels of each recombinant receptor and the effect of inverse agonist administration. Our data demonstrate that naturally occurring amino acid substitutions in the hD1R can lead to alterations in expression levels as well as in basal and ligand-induced signaling. The potency and efficacy of dopamine, synthetic agonists (i.e., fenoldopam, SKF-38393, SKF-82958, and SCH23390), and inverse agonists [i.e., flupenthixol and (+)butaclamol] were reduced at selected hD1R variants. Furthermore, inverse agonist induced effects on expression levels were sensitive to selected amino acid substitutions. In contrast to the hD1R variants, hD2R polymorphisms did not affect ligand function or receptor expression. The observation that the hD1R mutations induce significant alterations in pharmacologic properties may have implications both for disease susceptibility and/or therapeutic response to dopaminergic ligands.

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Cloning, expression and pharmacological characterisation of the mouse somatostatin sst5 receptor

Cited by 36 publications

References 21 publications

Comparison of functional profiles at human recombinant somatostatin sst₂ receptor: simultaneous determination of intracellular Ca²⁺ and luciferase expression in CHO‐K1 cells

Comparison of functional profiles at human recombinant somatostatin sst₂ receptor: simultaneous determination of intracellular Ca²⁺ and luciferase expression in CHO‐K1 cells

Point Mutations in Either Subunit of the GABA_BReceptor Confer Constitutive Activity to the Heterodimer

Pharmacological Analysis of Human D1 and D2 Dopamine Receptor Missense Variants

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Cloning, expression and pharmacological characterisation of the mouse somatostatin sst5 receptor

Cited by 36 publications

References 21 publications

Comparison of functional profiles at human recombinant somatostatin sst2 receptor: simultaneous determination of intracellular Ca2+ and luciferase expression in CHO‐K1 cells

Comparison of functional profiles at human recombinant somatostatin sst2 receptor: simultaneous determination of intracellular Ca2+ and luciferase expression in CHO‐K1 cells

Point Mutations in Either Subunit of the GABABReceptor Confer Constitutive Activity to the Heterodimer

Pharmacological Analysis of Human D1 and D2 Dopamine Receptor Missense Variants

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Comparison of functional profiles at human recombinant somatostatin sst₂ receptor: simultaneous determination of intracellular Ca²⁺ and luciferase expression in CHO‐K1 cells

Comparison of functional profiles at human recombinant somatostatin sst₂ receptor: simultaneous determination of intracellular Ca²⁺ and luciferase expression in CHO‐K1 cells

Point Mutations in Either Subunit of the GABA_BReceptor Confer Constitutive Activity to the Heterodimer