A Broad G Protein-Coupled Receptor Internalization Assay that Combines SNAP-Tag Labeling, Diffusion-Enhanced Resonance Energy Transfer, and a Highly Emissive Terbium Cryptate

Levoye, Angélique; Zwier, Jurriaan M.; Jaracz‐Ros, Agnieszka; Klipfel, Laurence; Cottet, Martin; Maurel, Damien; Bdioui, Sara; Balabanian, Karl; Prézeau, Laurent; Trinquet, Eric; Durroux, Thierry; Bachelerie, Françoise

doi:10.3389/fendo.2015.00167

Cited by 63 publications

(65 citation statements)

References 57 publications

(67 reference statements)

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“…The results of our study showed reduced internalization at 500 nM CYTL1 compared with 50 nM CYTL1. Reportedly, there was also reduced internalization extent at a higher concentration of ligand in other studies related with receptor internalization assays (36), which was similar with our results. In our study, it might be due to the fact that CYTL1 at high concentrations induced obvious receptor internalization at different time points and we detected robust internalization extent induced by 50 nM CYTL1 and modest internalization extent induced by 500 nM in 15 min.…”

Section: Discussionsupporting

confidence: 93%

Cytokine-like 1 Chemoattracts Monocytes/Macrophages via CCR2

Wang

et al. 2016

The Journal of Immunology

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Cytokine-like 1 (CYTL1) is a novel potential cytokine that was first identified in CD34+ cells derived from bone marrow and cord blood, and it was also found using our immunogenomics strategy. The immunobiological functions of CYTL1 remain largely unknown, and its potential receptor(s) has not been identified. A previous proposed hypothesis suggested that CYTL1 had structural similarities with CCL2 and that CCR2 was a potential receptor of CYTL1. In this study, we verify that CYTL1 possesses chemotactic activity and demonstrate that its functional receptor is CCR2B using a series of experiments performed in HEK293 cells expressing CCR2B or CCR2B-EGFP, including chemotaxis, receptor internalization, and radioactive binding assays. CYTL1 chemoattracts human monocytes but not PBLs, and its chemotactic activity toward monocytes is dependent on the CCR2B-ERK pathway. Furthermore, both human and mouse recombinant CYTL1 protein have chemotactic effects on macrophages from wild-type mice but not from Ccr2−/− mice. Furthermore, the chemotactic activity of CYTL1 is sensitive to pertussis toxin. All of the above data confirm that CCR2B is a functional receptor of CYTL1.

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Section: Discussionsupporting

confidence: 93%

Cytokine-like 1 Chemoattracts Monocytes/Macrophages via CCR2

Wang

et al. 2016

The Journal of Immunology

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“…In the present study, we show that an experimental setup in which labeling is performed at 4°C successfully detects constitutive internalization of GPRC6A. This is in agreement with Levoye et al (38) in which constitutive internalization of the CXC chemokine receptor 7 was detected by means of the real-time internalization assay using 4°C labeling.…”

Section: Discussionsupporting

confidence: 93%

“…The assay allows for detection of receptor internalization in a time-dependent manner and is performed in a 96-well format, thus enabling higher throughput than previously available methods. To verify the real-time internalization technique, two groups tested a range of GPCRs and correlated results from the realtime assay with classical immunocytochemistry experiments (37,38). Moreover, GLP1R internalization was tested in the presence of a dominant-negative mutant of dynamin, which blocks dynamin-dependent internalization.…”

Section: Discussionmentioning

confidence: 99%

“…A novel, real-time internalization assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) utilizes a Tag-lite SNAP Lumi4-Tb substrate for labeling of surface-expressed SNAP-tagged receptors with a donor fluorophore (37,38). After addition of excess amounts of a cell-impermeable acceptor fluorophore, internalization is allowed by adding buffer Ϯ agonist, and measurements of donor and acceptor emission are performed immediately after.…”

Section: A Novel Real-time Internalization Assay Confirmed the Constimentioning

confidence: 99%

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The GPRC6A receptor displays constitutive internalization and sorting to the slow recycling pathway

Jacobsen

Ammendrup-Johnsen

Jansen

et al. 2017

Journal of Biological Chemistry

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The class C G protein-coupled receptor GPRC6A is a putative nutrient-sensing receptor and represents a possible new drug target in metabolic disorders. However, the specific physiological role of this receptor has yet to be identified, and the mechanisms regulating its activity and cell surface availability also remain enigmatic. In the present study, we investigated the trafficking properties of GPRC6A by use of both a classical antibody feeding internalization assay in which cells were visualized using confocal microscopy and a novel internalization assay that is based on real-time measurements of fluorescence resonance energy transfer. Both assays revealed that GPRC6A predominantly undergoes constitutive internalization, whereas the agonist-induced effects were imperceptible. Moreover, postendocytic sorting was investigated by assessing the co-localization of internalized GPRC6A with selected Rab protein markers. Internalized GPRC6A was mainly co-localized with the early endosome marker Rab5 and the long loop recycling endosome marker Rab11 and to a much lesser extent with the late endosome marker Rab7. This suggests that upon agonist-independent internalization, GPRC6A is recycled via the Rab11-positive slow recycling pathway, which may be responsible for ensuring a persistent pool of GPRC6A receptors at the cell surface despite chronic agonist exposure. Distinct trafficking pathways have been reported for several of the class C receptors, and our results thus substantiate that non-canonical trafficking mechanisms are a common feature for the nutrient-sensing class C family that ensure functional receptors in the cell membrane despite prolonged agonist exposure.The G protein-coupled receptor, class C, group 6, member A (GPRC6A), 2 is a nutrient-sensing G protein-coupled receptor (GPCR) belonging to class C. It is activated by basic L-␣-amino acids and divalent cations, which trigger coupling to the G q protein and thereby an increase in the intracellular levels of the second messengers inositol trisphosphate and Ca 2ϩ (1-8). Other ligands and signaling pathways have been reported (3, 9 -13); however Rueda et al. (8) and we (7) have not been able to confirm these findings. The GPRC6A receptor displays a broad but low expression profile in human, mouse, and rat (2,5,10,11,14,15), thus giving little indication to the physiological role of the receptor. Several groups have conducted studies using GPRC6A knock-out mice to elucidate the physiological function of the receptor, and although results differ between knockout mouse models, they altogether suggest an involvement in metabolism and endocrine regulation (6, 10, 11, 16 -18).Over the years, it has become evident that GPCR signaling is much more complex than once believed. For most receptors, a variety of ligands and intracellular signaling pathways are available, and numerous regulatory mechanisms are thus required for obtaining specific biological responses (19).

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“…5.9, 5.12, and 6.3 of Stoddart et al (44)]. The intracellular FFAR2/3 fraction may be due to constitutive internalization of agonist-free GPCR, which has been observed for a subset of GPCRs, such as the putative nutrient sensing receptor GPRC6A (48) and the atypical chemokine receptor 3 (49). Confirming this will require further studies.…”

Section: Resultsmentioning

confidence: 98%

FFAR2‐FFAR3 receptor heteromerization modulates short‐chain fatty acid sensing

Ang

Xiong

et al. 2017

FASEB j.

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Free fatty acid receptors 2 and 3 (FFAR2/FFA2/GPR43 and FFAR3/FFA3/GPR41) are mammalian receptors for gut microbiota–derived short-chain fatty acids (SCFAs). These receptors are promising drug targets for obesity, colitis, colon cancer, asthma, and arthritis. Here, we demonstrate that FFAR2 and FFAR3 interact to form a heteromer in primary human monocytes and macrophages via proximity ligation assay, and during heterologous expression in HEK293 cells via bimolecular fluorescence complementation and fluorescence resonance energy transfer. The FFAR2-FFAR3 heteromer displayed enhanced cytosolic Ca2+ signaling (1.5-fold increase relative to homomeric FFAR2) and β-arrestin-2 recruitment (30-fold increase relative to homomeric FFAR3). The enhanced heteromer signaling was attenuated by FFAR2 antagonism (CATPB), Gαq inhibition (YM254890), or Gαi inhibition (pertussis toxin). Unlike homomeric FFAR2/3, the heteromer lacked the ability to inhibit cAMP production but gained the ability to induce p38 phosphorylation in HEK293 and inflammatory monocytes via a CATPB- and YM254890-sensitive mechanism. Our data, taken together, reveal that FFAR2 and FFAR3 may interact to form a receptor heteromer with signaling that is distinct from the parent homomers—a novel pathway for drug targeting.—Ang, Z., Xiong, D., Wu, M., Ding, J. L. FFAR2-FFAR3 receptor heteromerization modulates short-chain fatty acid sensing.

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A Broad G Protein-Coupled Receptor Internalization Assay that Combines SNAP-Tag Labeling, Diffusion-Enhanced Resonance Energy Transfer, and a Highly Emissive Terbium Cryptate

Cited by 63 publications

References 57 publications

Cytokine-like 1 Chemoattracts Monocytes/Macrophages via CCR2

Cytokine-like 1 Chemoattracts Monocytes/Macrophages via CCR2

The GPRC6A receptor displays constitutive internalization and sorting to the slow recycling pathway

FFAR2‐FFAR3 receptor heteromerization modulates short‐chain fatty acid sensing

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