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Vol. 289, Issue 2, 946-955, May 1999
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis,
Indiana
An assay for measuring agonist-stimulated
[35S]guanosine-5'-O-(3-thio)triphosphate
(GTP
35S) binding to heterotrimeric GTP binding
proteins was developed for use in 96-well format using commercially
available anti-G protein antibodies captured by anti-IgG-coated
scintillation proximity assay beads. Use of an anti-G
q/11 antibody
to measure GTP35S binding mediated by M1,
M3, and M5 receptors stably expressed in
Chinese hamster ovary (CHO) cells resulted in a marked increase in
agonist-stimulated/basal binding ratio compared with whole membrane
binding. Pertussis toxin (PTX) treatment of CHO M1 cells before membrane preparation resulted in a marked reduction in agonist-stimulated GTP35S binding to whole membranes.
Direct coupling of M1 receptors in CHO cells to inhibitory
G proteins was demonstrated using an anti-Gi(1-3) antibody, and
this binding was inhibited by 76% following PTX treatment. However,
PTX had no effect on M1-mediated binding determined using
anti-Gq/11. CHO M2 receptors mediated robust
agonist-stimulated GTP35S binding measured with
anti-Gi(1-3), but coupled only weakly to Gq/11. Using membranes
from rat striatum, GTP35S binding stimulated by
oxotremorine M was demonstrated using anti-Gq/11, anti-Gi(1-3),
and anti-Go antibodies. Agonist-stimulated binding to striatal
membranes showed a marked antibody-dependent GDP requirement with
robust signals obtained using 0.1 µM GDP for anti-Gq/11 compared
with 50 µM GDP for anti-Gi(1-3) and anti-Go. The potencies
observed for pirenzepine and AFDX 116 blockade of agonist-stimulated
GTP35S binding to striatal membranes determined with
anti-Gq/11 and anti-Go suggested mediation of these responses
primarily by M1 and M4 receptors, respectively.
Antibody capture GTP35S binding using scintillation
proximity assay technology provides a convenient, productive
alternative to immunoprecipitation for exploration of receptor-G
protein interaction in cells and tissues.
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