A luminescent assay for real-time measurements of receptor endocytosis in living cells.
Ligand-mediated endocytosis is a key autoregulatory mechanism governing the duration and intensity of signals emanating from cell surface receptors. Due to the mechanistic complexity of endocytosis and its emerging relevance in disease, simple methods capable of tracking this dynamic process in cells have become increasingly desirable. We have developed a bioluminescent reporter technology for real-time analysis of ligand-mediated receptor endocytosis using genetic fusions of NanoLuc luciferase with various G-protein-coupled receptors (GPCRs). This method is compatible with standard microplate formats, which should decrease work flows for high-throughput screens. This article also describes the application of this technology to endocytosis of epidermal growth factor receptor (EGFR), demonstrating potential applicability of the method beyond GPCRs.
Promega Corporation, Fitchburg, WI 53711, USA. Electronic address: matt.robers@promega.com.
Matthew B
MB
Robers
Promega Corporation, Fitchburg, WI 53711, USA.
Brock F
BF
Binkowski
Promega Corporation, Fitchburg, WI 53711, USA.
Mei
M
Cong
Promega Corporation, Fitchburg, WI 53711, USA.
Chad
C
Zimprich
Promega Biosciences, San Luis Obispo, CA 93401, USA.
Cesear
C
Corona
Promega Biosciences, San Luis Obispo, CA 93401, USA.
Mark
M
McDougall
Promega Corporation, Fitchburg, WI 53711, USA.
George
G
Otto
Promega Corporation, Fitchburg, WI 53711, USA.
Christopher T
CT
Eggers
Promega Corporation, Fitchburg, WI 53711, USA.
Jim
J
Hartnett
Promega Corporation, Fitchburg, WI 53711, USA.
Thomas
T
Machleidt
Promega Corporation, Fitchburg, WI 53711, USA.
Frank
F
Fan
Promega Corporation, Fitchburg, WI 53711, USA.
Keith V
KV
Wood
D000818:Animals; D060829:Arthropod Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D055808:Drug Discovery / Q000379:methods; D004705:Endocytosis / Q000187:drug effects; D005456:Fluorescent Dyes / Q000737:chemistry; D017930:Genes, Reporter / Q000187:drug effects; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D015850:Interleukin-6 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008156:Luciferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals / Q000187:drug effects; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D060829:Arthropod Proteins; D005456:Fluorescent Dyes; D015850:Interleukin-6; D008024:Ligands; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D008156:Luciferases
Endocytosis; G-protein-coupled receptor; Internalization; NanoLuc; Receptor tyrosine kinase