Postgenomic characterization of G-protein-coupled receptors

Mertens, Inge; Vandingenen, Anick; Meeusen, Tom; Loof, Arnold De; Schoofs, Liliane

doi:10.1517/14622416.5.6.657

Cited by 34 publications

(22 citation statements)

References 113 publications

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“…The resulting PCR product was directionally cloned into the eukaryotic expression vector pcDNA3.1D (Invitrogen) and sequenced. Human embryonic kidney (HEK293T) cells were transiently transfected with the Ce-GnRHR/pcDNA3.1 expression construct and the promiscuous G protein G␣16 using FuGene 6 (Roche) (37).…”

Section: Discussionmentioning

confidence: 99%

Adipokinetic hormone signaling through the gonadotropin-releasing hormone receptor modulates egg-laying in Caenorhabditis elegans

Lindemans

Liu

Janssen

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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In mammals, hypothalamic gonadotropin-releasing hormone (GnRH) is a neuropeptide that stimulates the release of gonadotropins from the anterior pituitary. The existence of a putative functional equivalent of this reproduction axis in protostomian invertebrates has been a matter of debate. In this study, the ligand for the GnRH receptor in the nematode Caenorhabditis elegans (Ce-GnRHR) was found using a bioinformatics approach. The peptide and its precursor are reminiscent of both insect adipokinetic hormones and GnRH-preprohormone precursors from tunicates and higher vertebrates. We cloned the AKH-GnRH-like preprohormone and the Ce-GnRHR and expressed the GPCR in HEK293T cells. The GnRHR was activated by the C. elegans AKH-GnRH-like peptide (EC50 ‫؍‬ 150 nM) and by Drosophila AKH and other nematode AKH-GnRHs that we found in EST databases. Analogous to both insect AKH receptor and vertebrate GnRH receptor signaling, Ce-AKH-GnRH activated its receptor through a G␣q protein with Ca 2؉ as a second messenger. Gene silencing of Ce-GnRHR, Ce-AKHGnRH, or both resulted in a delay in the egg-laying process, comparable to a delay in puberty in mammals lacking a normal dose of GnRH peptide or with a mutated GnRH precursor or receptor gene. The present data support the view that the AKH-GnRH signaling system probably arose very early in metazoan evolution and that its role in reproduction might have been developed before the divergence of protostomians and deuterostomians.C. elegans ͉ G protein-coupled receptor ͉ molecular evolution ͉ neuropeptide ͉ neuroendocrinology

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Section: Discussionmentioning

confidence: 99%

Adipokinetic hormone signaling through the gonadotropin-releasing hormone receptor modulates egg-laying in Caenorhabditis elegans

Lindemans

Liu

Janssen

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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140

136

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“…The effects of PDF are mediated by a single GPCR, the Drosophila PDFR, a member of the Secretin receptor (B1) family most closely related to mammalian receptors for calcitonin and calcitonin gene-related peptide (Hewes and Taghert, 2001;Mertens et al, 2004;Hyun et al, 2005;Lear et al, 2005). Interestingly, PDFR is not expressed in all clock neurons (Fig.…”

Section: Animal Models To Discover and Validate Molecular Conceptsmentioning

confidence: 99%

Model Organisms in G Protein–Coupled Receptor Research

et al. 2015

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The study of G protein-coupled receptors (GPCRs) has benefited greatly from experimental approaches that interrogate their functions in controlled, artificial environments. Working in vitro, GPCR receptorologists discovered the basic biologic mechanisms by which GPCRs operate, including their eponymous capacity to couple to G proteins; their molecular makeup, including the famed serpentine transmembrane unit; and ultimately, their three-dimensional structure. Although the insights gained from working outside the native environments of GPCRs have allowed for the collection of low-noise data, such approaches cannot directly address a receptor's native (in vivo) functions. An in vivo approach can complement the rigor of in vitro approaches: as studied in model organisms, it imposes physiologic constraints on receptor action and thus allows investigators to deduce the most salient features of receptor function. Here, we briefly discuss specific examples in which model organisms have successfully contributed to the elucidation of signals controlled through GPCRs and other surface receptor systems. We list recent examples that have served either in the initial discovery of GPCR signaling concepts or in their fuller definition. Furthermore, we selectively highlight experimental advantages, shortcomings, and tools of each model organism.

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“…The GPCR of interest is therefore cloned and subsequently transfected in a cellular expression system. In the most commonly used methods, receptor activation is determined by measuring changes in the concentration of second messenger molecules 9 . The main receptor screening assays rely on calcium-sensitive bioluminescent proteins (e.g., aequorin) 10 or fluorescent calcium indicators (e.g., Fluo-4)…”

Section: Introductionmentioning

confidence: 99%

“…The fluorescence-based assays, in which receptor-expressing cells are loaded with a fluorescent calcium indicator prior to ligand screening, have the advantage that they allow high-throughput screening due to their ease of use, short reading time, and the flexibility of screening multiple orphan receptors on a single plate . The availability of medium/high-throughput screening approaches, like the fluorescence-based calcium mobilization assay that has proven to be a widely applied technique 9,18 , is therefore invaluable.…”

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confidence: 99%

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

Caers

Peymen

Suetens

et al. 2014

JoVE

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For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G proteincoupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous Gα 16 construct is co-transfected. Following ligand binding, activation of the Gα 16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous Gα 16 , which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

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Postgenomic characterization of G-protein-coupled receptors

Cited by 34 publications

References 113 publications

Adipokinetic hormone signaling through the gonadotropin-releasing hormone receptor modulates egg-laying in Caenorhabditis elegans

Adipokinetic hormone signaling through the gonadotropin-releasing hormone receptor modulates egg-laying in Caenorhabditis elegans

Model Organisms in G Protein–Coupled Receptor Research

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

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