The receptor-stimulated accumulation of [S-35]GTP gammaS provides a measure
of functional coupling of G proteins with receptors. Sensitivity for autor
adiographic visualization of [S-35]GTP gammaS binding was improved two- to
threefold in rat brain sections by optimizing assay conditions. Non-specifi
c (NSB). basal and agonist-stimulated [S-35]GTP gammaS binding were measure
d, using methadone, 5-carboxamidotryptamine and epinephrine for mu -opiate
receptors, 5-HT1A receptors and alpha (2)-adrenoceptors. Basal and NSB were
low in glycylglycine buffer compared to Tris or HEPES buffers, and agonist
-stimulated [S-35]GTP gammaS binding was more easily observed. Further opti
mization using glycylglycine buffer found increased signal-to-noise ratio w
ith inclusion of dithiothrietol, increased [S-35]GTP gammaS incubation time
(2-4 h) and guanosine 5'-diphosphate (GDP) preincubation (20-30 min), and
use of [S-35]GTP gammaS at 0.1 nM. Improved sensitivity was due to decrease
d NSB and basal [S-35]GTP gammaS binding and agonist-stimulated binding wer
e similarly affected for each receptor system. The assay conditions describ
ed should extend the use of agonist-stimulated [S-35]GTP gammaS autoradiogr
aphy to receptors, which produce low levels of [S-35]GTP gammaS binding and
to the measurement of changes in receptor-G protein coupling. (C) 2001 Pub
lished by Elsevier Science B.V.