dc.contributor.author |
Kumar, Shree Senthil |
|
dc.contributor.author |
Ward, Marie-Louise |
|
dc.contributor.author |
Mountjoy, Kathleen Grace |
|
dc.coverage.spatial |
England |
|
dc.date.accessioned |
2022-03-03T01:18:10Z |
|
dc.date.available |
2022-03-03T01:18:10Z |
|
dc.date.issued |
2021-4-26 |
|
dc.identifier.citation |
Journal of molecular endocrinology 66(4):285-297 26 Apr 2021 |
|
dc.identifier.issn |
0952-5041 |
|
dc.identifier.uri |
https://hdl.handle.net/2292/58370 |
|
dc.description.abstract |
The melanocortin-4 receptor (MC4R), a critical G-protein-coupled receptor (GPCR) regulating energy homeostasis, activates multiple signalling pathways, including mobilisation of intracellular calcium ([Ca2+]i). However, very little is known about the physiological significance of MC4R-induced [Ca2+]i since few studies measure MC4R-induced [Ca2+]i. High-throughput, read-out assays for [Ca2+]i have proven unreliable for overexpressed GPCRs like MC4R, which exhibit low sensitivity mobilising [Ca2+]i. Therefore, we developed, optimised, and validated a robust quantitative high-throughput assay using Fura-2 ratio-metric calcium dye and HEK293 cells stably transfected with MC4R. The quantitation enables direct comparisons between assays and even between different research laboratories. Assay conditions were optimised step-by-step to eliminate interference from stretch-activated receptor increases in [Ca2+]i and to maximise ligand-activated MC4R-induced [Ca2+]i. Calcium imaging was performed using a PheraStar FS multi-well plate reader. Probenecid, included in the buffers to prevent extrusion of Fura-2 dye from cells, was found to interfere with the EGTA-chelation of calcium, required to determine Rmin for quantitation of [Ca2+]i. Therefore, we developed a method to determine Rmin in specific wells without probenecid, which was run in parallel with each assay. The validation of the assay was shown by reproducible α-melanocyte-stimulating hormone (α-MSH) concentration-dependent activation of the stably expressed human MC4R (hMC4R) and mouse MC4R (mMC4R), inducing increases in [Ca2+]i, for three independent experiments. This robust, reproducible, high-throughput assay that quantitatively measures MC4R-induced mobilisation of [Ca2+]i in vitro has potential to advance the development of therapeutic drugs and understanding of MC4R signalling associated with human obesity. |
|
dc.format.medium |
Electronic |
|
dc.language |
eng |
|
dc.publisher |
Bioscientifica |
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dc.relation.ispartofseries |
Journal of molecular endocrinology |
|
dc.rights |
Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. Previously published items are made available in accordance with the copyright policy of the publisher. |
|
dc.rights.uri |
https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm |
|
dc.rights.uri |
https://creativecommons.org/licenses/by/4.0/ |
|
dc.subject |
Humans |
|
dc.subject |
Calcium |
|
dc.subject |
Receptor, Melanocortin, Type 4 |
|
dc.subject |
Cyclic AMP |
|
dc.subject |
Signal Transduction |
|
dc.subject |
Calcium Signaling |
|
dc.subject |
Amino Acid Sequence |
|
dc.subject |
Protein Binding |
|
dc.subject |
Energy Metabolism |
|
dc.subject |
Homeostasis |
|
dc.subject |
High-Throughput Screening Assays |
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dc.subject |
HEK293 Cells |
|
dc.subject |
G-protein-coupled receptor |
|
dc.subject |
calcium |
|
dc.subject |
cell signalling |
|
dc.subject |
high-throughput screening |
|
dc.subject |
melanocortin-4 receptor |
|
dc.subject |
Amino Acid Sequence |
|
dc.subject |
Calcium |
|
dc.subject |
Calcium Signaling |
|
dc.subject |
Cyclic AMP |
|
dc.subject |
Energy Metabolism |
|
dc.subject |
HEK293 Cells |
|
dc.subject |
High-Throughput Screening Assays |
|
dc.subject |
Homeostasis |
|
dc.subject |
Humans |
|
dc.subject |
Protein Binding |
|
dc.subject |
Receptor, Melanocortin, Type 4 |
|
dc.subject |
Signal Transduction |
|
dc.subject |
Science & Technology |
|
dc.subject |
Life Sciences & Biomedicine |
|
dc.subject |
Endocrinology & Metabolism |
|
dc.subject |
melanocortin-4 receptor |
|
dc.subject |
cell signalling |
|
dc.subject |
calcium |
|
dc.subject |
G-protein-coupled receptor |
|
dc.subject |
high-throughput screening |
|
dc.subject |
MITOGEN-ACTIVATED PROTEIN |
|
dc.subject |
ENDOTHELIAL-CELLS |
|
dc.subject |
IN-VITRO |
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dc.subject |
FURA-2 |
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dc.subject |
CA2+ |
|
dc.subject |
SEQUESTRATION |
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dc.subject |
MOBILIZATION |
|
dc.subject |
PHARMACOLOGY |
|
dc.subject |
INHIBITION |
|
dc.subject |
MUTATIONS |
|
dc.subject |
0707 Veterinary Sciences |
|
dc.subject |
1103 Clinical Sciences |
|
dc.subject |
1114 Paediatrics and Reproductive Medicine |
|
dc.title |
Quantitative high-throughput assay to measure MC4R-induced intracellular calcium. |
|
dc.type |
Journal Article |
|
dc.identifier.doi |
10.1530/jme-20-0285 |
|
pubs.issue |
4 |
|
pubs.begin-page |
285 |
|
pubs.volume |
66 |
|
dc.date.updated |
2022-02-16T01:37:46Z |
|
dc.rights.holder |
Copyright: The author |
en |
pubs.author-url |
https://www.ncbi.nlm.nih.gov/pubmed/33739935 |
|
pubs.end-page |
297 |
|
pubs.publication-status |
Published |
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dc.rights.accessrights |
http://purl.org/eprint/accessRights/OpenAccess |
en |
pubs.subtype |
Research Support, Non-U.S. Gov't |
|
pubs.subtype |
research-article |
|
pubs.subtype |
Journal Article |
|
pubs.elements-id |
845553 |
|
dc.identifier.eissn |
1479-6813 |
|
dc.identifier.pii |
JME-20-0285 |
|