A pH-Sensitive Fluor, CypHerTM 5, Used to Monitor Agonist-Induced G Protein-Coupled Receptor Internalization in Live Cells
G protein-coupled receptors (GPCRs) are the largest family of proteins involved in transmembrane signal transduction and are actively studied because of their suitability as therapeutic small-molecule drug targets. Agonist activation of GPCRs almost invariably results in the receptor being desensiti...
Saved in:
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Taylor & Francis Group
2002-11-01
|
| Series: | BioTechniques |
| Online Access: | https://www.future-science.com/doi/10.2144/02335dd10 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850152110734180352 |
|---|---|
| author | E.J. Adie S. Kalinka L. Smith M.J. Francis A. Marenghi M.E. Cooper M. Briggs N.P. Michael G. Milligan S. Game |
| author_facet | E.J. Adie S. Kalinka L. Smith M.J. Francis A. Marenghi M.E. Cooper M. Briggs N.P. Michael G. Milligan S. Game |
| author_sort | E.J. Adie |
| collection | DOAJ |
| description | G protein-coupled receptors (GPCRs) are the largest family of proteins involved in transmembrane signal transduction and are actively studied because of their suitability as therapeutic small-molecule drug targets. Agonist activation of GPCRs almost invariably results in the receptor being desensitized. One of the key events in receptor desensitization is the sequestration of the receptor from the cell surface into acidic intracellular endosomes. Therefore, a convenient, generic, and noninvasive monitor of this process is desirable. A novel, pH-sensitive, red-excited fluorescent dye, CypHerTM 5, was synthesized. This dye is non-fluorescent at neutral pH and is fluorescent at acidic pH. Anti-epitope antibodies labeled with this dye were internalized in an agonist concentration- and time-dependent manner, following binding on live cells to a range of GPCRs that had been modified to incorporate the epitope tags in their extracellular N-terminal domain. This resulted in a large signal increase over background. When protonated, the red fluorescence of CypHer 5 provides a generic reagent suitable for monitoring the internalization of GPCRs into acidic vesicles. This approach should be amenable to the study of many other classes of cell surface receptors that also internalize following stimulation. |
| format | Article |
| id | doaj-art-2dcd98267e8e469e94423d7a548fa3a9 |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2002-11-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-2dcd98267e8e469e94423d7a548fa3a92025-08-20T02:26:03ZengTaylor & Francis GroupBioTechniques0736-62051940-98182002-11-013351152115710.2144/02335dd10A pH-Sensitive Fluor, CypHerTM 5, Used to Monitor Agonist-Induced G Protein-Coupled Receptor Internalization in Live CellsE.J. Adie0S. Kalinka1L. Smith2M.J. Francis3A. Marenghi4M.E. Cooper5M. Briggs6N.P. Michael7G. Milligan8S. Game91Amersham Biosciences, Cardiff, UK1Amersham Biosciences, Cardiff, UK1Amersham Biosciences, Cardiff, UK1Amersham Biosciences, Cardiff, UK1Amersham Biosciences, Cardiff, UK1Amersham Biosciences, Cardiff, UK1Amersham Biosciences, Cardiff, UK1Amersham Biosciences, Cardiff, UK1Amersham Biosciences, Cardiff, UK1Amersham Biosciences, Cardiff, UKG protein-coupled receptors (GPCRs) are the largest family of proteins involved in transmembrane signal transduction and are actively studied because of their suitability as therapeutic small-molecule drug targets. Agonist activation of GPCRs almost invariably results in the receptor being desensitized. One of the key events in receptor desensitization is the sequestration of the receptor from the cell surface into acidic intracellular endosomes. Therefore, a convenient, generic, and noninvasive monitor of this process is desirable. A novel, pH-sensitive, red-excited fluorescent dye, CypHerTM 5, was synthesized. This dye is non-fluorescent at neutral pH and is fluorescent at acidic pH. Anti-epitope antibodies labeled with this dye were internalized in an agonist concentration- and time-dependent manner, following binding on live cells to a range of GPCRs that had been modified to incorporate the epitope tags in their extracellular N-terminal domain. This resulted in a large signal increase over background. When protonated, the red fluorescence of CypHer 5 provides a generic reagent suitable for monitoring the internalization of GPCRs into acidic vesicles. This approach should be amenable to the study of many other classes of cell surface receptors that also internalize following stimulation.https://www.future-science.com/doi/10.2144/02335dd10 |
| spellingShingle | E.J. Adie S. Kalinka L. Smith M.J. Francis A. Marenghi M.E. Cooper M. Briggs N.P. Michael G. Milligan S. Game A pH-Sensitive Fluor, CypHerTM 5, Used to Monitor Agonist-Induced G Protein-Coupled Receptor Internalization in Live Cells BioTechniques |
| title | A pH-Sensitive Fluor, CypHerTM 5, Used to Monitor Agonist-Induced G Protein-Coupled Receptor Internalization in Live Cells |
| title_full | A pH-Sensitive Fluor, CypHerTM 5, Used to Monitor Agonist-Induced G Protein-Coupled Receptor Internalization in Live Cells |
| title_fullStr | A pH-Sensitive Fluor, CypHerTM 5, Used to Monitor Agonist-Induced G Protein-Coupled Receptor Internalization in Live Cells |
| title_full_unstemmed | A pH-Sensitive Fluor, CypHerTM 5, Used to Monitor Agonist-Induced G Protein-Coupled Receptor Internalization in Live Cells |
| title_short | A pH-Sensitive Fluor, CypHerTM 5, Used to Monitor Agonist-Induced G Protein-Coupled Receptor Internalization in Live Cells |
| title_sort | ph sensitive fluor cyphertm 5 used to monitor agonist induced g protein coupled receptor internalization in live cells |
| url | https://www.future-science.com/doi/10.2144/02335dd10 |
| work_keys_str_mv | AT ejadie aphsensitivefluorcyphertm5usedtomonitoragonistinducedgproteincoupledreceptorinternalizationinlivecells AT skalinka aphsensitivefluorcyphertm5usedtomonitoragonistinducedgproteincoupledreceptorinternalizationinlivecells AT lsmith aphsensitivefluorcyphertm5usedtomonitoragonistinducedgproteincoupledreceptorinternalizationinlivecells AT mjfrancis aphsensitivefluorcyphertm5usedtomonitoragonistinducedgproteincoupledreceptorinternalizationinlivecells AT amarenghi aphsensitivefluorcyphertm5usedtomonitoragonistinducedgproteincoupledreceptorinternalizationinlivecells AT mecooper aphsensitivefluorcyphertm5usedtomonitoragonistinducedgproteincoupledreceptorinternalizationinlivecells AT mbriggs aphsensitivefluorcyphertm5usedtomonitoragonistinducedgproteincoupledreceptorinternalizationinlivecells AT npmichael aphsensitivefluorcyphertm5usedtomonitoragonistinducedgproteincoupledreceptorinternalizationinlivecells AT gmilligan aphsensitivefluorcyphertm5usedtomonitoragonistinducedgproteincoupledreceptorinternalizationinlivecells AT sgame aphsensitivefluorcyphertm5usedtomonitoragonistinducedgproteincoupledreceptorinternalizationinlivecells AT ejadie phsensitivefluorcyphertm5usedtomonitoragonistinducedgproteincoupledreceptorinternalizationinlivecells AT skalinka phsensitivefluorcyphertm5usedtomonitoragonistinducedgproteincoupledreceptorinternalizationinlivecells AT lsmith phsensitivefluorcyphertm5usedtomonitoragonistinducedgproteincoupledreceptorinternalizationinlivecells AT mjfrancis phsensitivefluorcyphertm5usedtomonitoragonistinducedgproteincoupledreceptorinternalizationinlivecells AT amarenghi phsensitivefluorcyphertm5usedtomonitoragonistinducedgproteincoupledreceptorinternalizationinlivecells AT mecooper phsensitivefluorcyphertm5usedtomonitoragonistinducedgproteincoupledreceptorinternalizationinlivecells AT mbriggs phsensitivefluorcyphertm5usedtomonitoragonistinducedgproteincoupledreceptorinternalizationinlivecells AT npmichael phsensitivefluorcyphertm5usedtomonitoragonistinducedgproteincoupledreceptorinternalizationinlivecells AT gmilligan phsensitivefluorcyphertm5usedtomonitoragonistinducedgproteincoupledreceptorinternalizationinlivecells AT sgame phsensitivefluorcyphertm5usedtomonitoragonistinducedgproteincoupledreceptorinternalizationinlivecells |