PMID- 20103692
OWN - NLM
STAT- MEDLINE
DCOM- 20100628
LR  - 20110523
IS  - 1552-454X (Electronic)
IS  - 1087-0571 (Linking)
VI  - 15
IP  - 3
DP  - 2010 Mar
TI  - Homogeneous GTP binding assay employing QRET technology.
PG  - 261-7
LID - 10.1177/1087057109358921 [doi]
AB  - Functional cell signaling assays have become important tools for measuring 
      ligand-induced receptor activation in cell-based biomolecular screening. 
      Guanosine-5'-triphosphate (GTP) is a generic signaling marker responsible for the 
      first intracellular signaling event of the G-protein-coupled receptors (GPCRs). 
      [(35)S]GTPgammaS binding assay is the classical well-established method for 
      measuring agonist-induced G-protein activation requiring a separation of free and 
      bound fractions prior to measurement. Here a novel, separation-free, 
      time-resolved fluorescence GTP binding assay has been developed based on a 
      non-fluorescence resonance energy transfer (FRET) single-label approach and 
      quenching of a nonbound europium-labeled, nonhydrolyzable GTP analog (Eu-GTP). 
      The quenching resonance energy transfer (QRET) method relies on the use of 
      Eu-GTP, providing a time-resolved fluorescent detection as an alternative to the 
      radiolabel [(35)S]GTPgammaS assay. Upon activation of recombinant human 
      alpha(2A)-adrenoceptors (alpha(2A)-AR) expressed in Chinese hamster ovary cells, 
      guanosine-5'-diphosphate is released from the alpha-subunit of Gi-proteins, 
      enabling the subsequent binding of Eu-GTP. Activation of alpha(2A)-AR with 5 
      different alpha(2)-AR agonists was measured quantitatively using the developed 
      QRET GTP assay and compared to [(35)S]GTPgammaS and heterogeneous Eu-GTP 
      filtration assays. Equal potencies and efficacy rank orders were observed in all 
      3 assays but with a lower signal-to-background ratio and increased assay 
      variation in the QRET assay compared to the Eu-GTP filtration and the 
      nonhomogeneous [(35)S]GTPgammaS binding assays.
FAU - Rozwandowicz-Jansen, Anita
AU  - Rozwandowicz-Jansen A
AD  - Laboratory of Biophysics, Institute of Biomedicine, University of Turku, Finland. 
      aniroz@utu.fi
FAU - Laurila, Jonne
AU  - Laurila J
FAU - Martikkala, Eija
AU  - Martikkala E
FAU - Frang, Heini
AU  - Frang H
FAU - Hemmila, Ilkka
AU  - Hemmila I
FAU - Scheinin, Mika
AU  - Scheinin M
FAU - Hanninen, Pekka
AU  - Hanninen P
FAU - Harma, Harri
AU  - Harma H
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20100126
PL  - United States
TA  - J Biomol Screen
JT  - Journal of biomolecular screening
JID - 9612112
RN  - 0 (Membrane Proteins)
RN  - 37589-80-3 (Guanosine 5'-O-(3-Thiotriphosphate))
RN  - 86-01-1 (Guanosine Triphosphate)
SB  - IM
MH  - Animals
MH  - Biological Assay/*methods
MH  - CHO Cells
MH  - Cricetinae
MH  - Cricetulus
MH  - *Energy Transfer
MH  - Fluorescence
MH  - Guanosine 5'-O-(3-Thiotriphosphate)/metabolism
MH  - Guanosine Triphosphate/*metabolism
MH  - Humans
MH  - Membrane Proteins/metabolism
MH  - Time Factors
EDAT- 2010/01/28 06:00
MHDA- 2010/06/29 06:00
CRDT- 2010/01/28 06:00
PHST- 2010/01/28 06:00 [entrez]
PHST- 2010/01/28 06:00 [pubmed]
PHST- 2010/06/29 06:00 [medline]
AID - 1087057109358921 [pii]
AID - 10.1177/1087057109358921 [doi]
PST - ppublish
SO  - J Biomol Screen. 2010 Mar;15(3):261-7. doi: 10.1177/1087057109358921. Epub 2010 
      Jan 26.