Chimeric G Proteins Allow a High-Throughput Signaling Assay of Gi-Coupled Receptors

Coward, Peter; Chan, S. D. H.; Wada, Hiroshi; Humphries, Gillian M. K.; Conklin, Bruce R.

doi:10.1006/abio.1999.4061

Cited by 214 publications

(162 citation statements)

References 31 publications

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“…The data obtained for the stable clones are consistent with the importance of the ratio of GPCR to G protein for efficiency of coupling to the functional response. Similarly, other reports have demonstrated that some GPCRs, for example selected opioid receptors, couple more efficiently to chimeric G proteins than to G␣ 16 (24). The CRE reporter gene assay data presented confirm the high potency of NPAF and NPFF peptides for HLWAR77 and demonstrate receptor coupling to native G i/o ␣-G protein subunits.…”

Section: Resultssupporting

confidence: 83%

“…Our initial activities were dependent on HL-WAR77 co-expression with G␣ 16 . Subsequently, we co-expressed HLWAR77 with a variety of other reported promiscuous G proteins and the "chimeric" G protein ␣ subunits (22)(23)(24), composed of ␣ q in which the five carboxyl-terminal residues are replaced with the carboxyl-terminal residues derived from ␣ i2 or ␣ o , referred to as G qi5 or G qo5 , respectively. Co-transfection of HLWAR77 with these chimeras resulted in a 3-4-fold enhancement of the calcium mobilization signal compared with the amplitude of the response obtained when the receptor was co-expressed with G␣ 16 (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Receptor for the Pain Modulatory Neuropeptides FF and AF Is an Orphan G Protein-coupled Receptor

Elshourbagy

Ames

Fitzgerald

et al. 2000

Journal of Biological Chemistry

241

172

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Opiate tolerance and dependence are major clinical and social problems. The anti-opiate neuropeptides FF and AF (NPFF and NPAF) have been implicated in pain modulation as well as in opioid tolerance and may play a critical role in this process, although their mechanism of action has remained unknown. Here we describe a cDNA encoding a novel neuropeptide Y-like human orphan G protein-coupled receptor (GPCR), referred to as HLWAR77 for which NPAF and NPFF have high affinity. Cells transiently or stably expressing HLWAR77 bind and respond in a concentration-dependent manner to NPAF and NPFF and are also weakly activated by FMRF-amide (Phe-Met-Arg-Phe-amide) and a variety of related peptides. The high affinity and potency of human NPFF and human NPAF for HLWAR77 strongly suggest that these are the cognate ligands for this receptor. Expression of HLWAR77 was demonstrated in brain regions associated with opiate activity, consistent with the pain-modulating activity of these peptides, whereas the expression in adipose tissue suggests other physiological and pathophysiological activities for FMRF-amide neuropeptides. The discovery that the anti-opiate neuropeptides are the endogenous ligands for HL-WAR77 will aid in defining the physiological role(s) of these ligands and facilitate the identification of receptor agonists and antagonists.

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Section: Resultssupporting

confidence: 83%

Section: Resultsmentioning

confidence: 99%

Receptor for the Pain Modulatory Neuropeptides FF and AF Is an Orphan G Protein-coupled Receptor

Elshourbagy

Ames

Fitzgerald

et al. 2000

Journal of Biological Chemistry

241

172

View full text Add to dashboard Cite

show abstract

“…The chimeric G qi5 protein, whose last five amino acid residues in the C-terminus was replaced with the corresponding portion of G i protein, is useful for monitoring the responses mediated by stimulation of G i / o protein-coupled receptors in the Xenopus oocyte expression system, as reported previously by our laboratory (10,11). Chimeric G qi5 allows G i / o -coupled receptors to couple to the phospholipase C (PLC)-mediated signal pathway (12,13). Agonists for G i / o protein-coupled receptors can elicit Ca 2+ -activated Cl − currents in such oocytes only through the chimeric G qi5 , but not through G i / o endogenously expressed in oocytes (10,11).…”

Section: Introductionmentioning

confidence: 94%

μ-Opioid Receptor Forms a Functional Heterodimer With Cannabinoid CB1 Receptor: Electrophysiological and FRET Assay Analysis

et al. 2008

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Abstract. Interactions between μ-opioid receptor (μOR) and cannabinoid CB 1 receptor (CB 1 R) were examined by morphological and electrophysiological methods. In baby hamster kidney (BHK) cells coexpressing μOR fused to the yellow fluorescent protein Venus and CB 1 R fused to the cyan fluorescent protein Cerulean, both colors were detected on the cell surface; and fluorescence resonance energy transfer (FRET) analysis revealed that μOR and CB 1 R formed a heterodimer. Coimmunoprecipitation and Western blotting analyses also confirmed the heterodimers of μOR and CB 1 R. [D-Ala 2 ,N-Me-Phe 4 ,Gly 5 -ol]enkephalin (DAMGO) or CP55,940 elicited K + currents in Xenopus oocytes expressing μOR or CB 1 R together with G protein activated-inwardly rectifying K + channels (GIRKs), respectively. In oocytes coexpressing both receptors, either of which was fused to the chimeric Gα protein G qi5 that activates the phospholipase C pathway, both DAMGO and CP55,940 elicited Ca 2+ -activated Cl − currents, indicating that each agonist can induce responses through G qi5 fused to either its own receptor or the other. Experiments with endogenous G i/o protein inactivation by pertussis toxin (PTX) supported the functional heterodimerization of μOR/ CB 1 R through PTX-insensitive G qi5(m) fused to each receptor. Thus, μOR and CB 1 R form a heterodimer and transmit a signal through a common G protein. Our electrophysiological method could be useful for determination of signals mediated through heterodimerized G protein-coupled receptors.

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“…Blood samples were withdrawn at various times, and plasma was stored frozen until analyzed. Plasma samples were analyzed either by HPLC using ultraviolet detection methods as described previously (13).…”

mentioning

confidence: 99%

A chemokine receptor CCR-1 antagonist reduces renal fibrosis after unilateral ureter ligation

Anders¹,

Vielhauer²,

Frink³

et al. 2002

J. Clin. Invest.

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Chimeric G Proteins Allow a High-Throughput Signaling Assay of Gi-Coupled Receptors

Cited by 214 publications

References 31 publications

Receptor for the Pain Modulatory Neuropeptides FF and AF Is an Orphan G Protein-coupled Receptor

Receptor for the Pain Modulatory Neuropeptides FF and AF Is an Orphan G Protein-coupled Receptor

μ-Opioid Receptor Forms a Functional Heterodimer With Cannabinoid CB1 Receptor: Electrophysiological and FRET Assay Analysis

A chemokine receptor CCR-1 antagonist reduces renal fibrosis after unilateral ureter ligation

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