PMID- 26179037
OWN - NLM
STAT- MEDLINE
DCOM- 20160406
LR  - 20161125
IS  - 1940-6029 (Electronic)
IS  - 1064-3745 (Linking)
VI  - 1322
DP  - 2015
TI  - Evaluating the Activity of Smoothened Toward G Proteins Using [(3)(5)S]Guanosine 
      5'-(3-O-thio)triphosphate ([(3)(5)S]GTPgammaS).
PG  - 35-44
LID - 10.1007/978-1-4939-2772-2_4 [doi]
AB  - The utilization of heterotrimeric G protein, and in particular those of the Gi, 
      family, by Hedgehogs through Smoothened has become increasingly clear. We 
      describe here a method for evaluating the activity of Smoothened toward G 
      proteins in membranes derived from human embryonic kidney-293 (HEK293) cells. The 
      assay relies on receptor-promoted exchange of GDP for [(35)S]GTPgammaS on the Galpha 
      subunit. The assay is best suited for analysis of the constitutive activity of 
      Smoothened, inverse agonism superimposed on this activity, and neutral antagonism 
      superimposed on inverse agonism. The assay would also be suitable for several 
      other applications requiring a proximal, quantifiable readout of Smoothened 
      activity.
FAU - Manning, David R
AU  - Manning DR
AD  - Department of Pharmacology, Perelman School of Medicine, University of 
      Pennsylvania, 3620 Hamilton Walk, Pennsylvania, PA, 19104-6084, USA, 
      manning@mail.med.upenn.edu.
FAU - Shen, Feng
AU  - Shen F
FAU - Riobo, Natalia A
AU  - Riobo NA
LA  - eng
GR  - R01 GM080396/GM/NIGMS NIH HHS/United States
GR  - GM080396/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PL  - United States
TA  - Methods Mol Biol
JT  - Methods in molecular biology (Clifton, N.J.)
JID - 9214969
RN  - 0 (Receptors, G-Protein-Coupled)
RN  - 0 (SMO protein, human)
RN  - 0 (Smoothened Receptor)
RN  - 0 (Sulfur Radioisotopes)
RN  - 37589-80-3 (Guanosine 5'-O-(3-Thiotriphosphate))
RN  - EC 3.6.1.- (GTP-Binding Proteins)
SB  - IM
MH  - GTP-Binding Proteins/chemistry/*metabolism
MH  - *Guanosine 5'-O-(3-Thiotriphosphate)/chemistry
MH  - HEK293 Cells
MH  - Humans
MH  - Immunoprecipitation/methods
MH  - Protein Binding
MH  - Receptors, G-Protein-Coupled/chemistry/*metabolism
MH  - Smoothened Receptor
MH  - Sulfur Radioisotopes/chemistry
EDAT- 2015/07/17 06:00
MHDA- 2016/04/07 06:00
CRDT- 2015/07/17 06:00
PHST- 2015/07/17 06:00 [entrez]
PHST- 2015/07/17 06:00 [pubmed]
PHST- 2016/04/07 06:00 [medline]
AID - 10.1007/978-1-4939-2772-2_4 [doi]
PST - ppublish
SO  - Methods Mol Biol. 2015;1322:35-44. doi: 10.1007/978-1-4939-2772-2_4.