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Fig. 5. GPR-1/2 is a GDI for GPA-16; RIC-8 is not a GEF for GPA-16. (A,B,E) Surface
plasmon resonance was used to analyze the binding of GPA-16 to GST-GPR-1/2
(amino acids 374-476) (A) and to GST-RIC-8 (B), as well as of GOA-1 to
GST-RIC-8 (E). GST fusion proteins were immobilized on a GST antibody
biosensor surface. `Analyte' (30 µl of 2 µM GPA-16 or GOA-1), in each of
the indicated nucleotide bound states, was injected over the biosensor
surface. Non-specific binding to GST was subtracted from each curve. (C)
Time-course of [35S]GTP
S binding to 100 nM GPA-16 in the
presence or absence of 10 µM GPR-1/2 peptide (amino acids 423-461). Results
are the mean±s.e.m. of duplicate samples. Observed association rate
constants (with 95% confidence intervals in parentheses) were: GPA-16, 0.73
(0.44-1.0) minutes-1; GPA-16+GPR-1/2, 0.069 (0.050-0.087)
minutes-1. (D,F) Time-course of [35S]GTPS binding
to 100 nM GPA-16 (D) or 100 nM GOA-1 (F) in the presence or absence of 2 µM
RIC-8 (D) or 200 nM RIC-8 (F). Results are the mean±s.e.m. of duplicate
samples. Observed association rate constants (with 95% confidence intervals in
parentheses) were: GPA-16, 0.32 (0.24-0.40) minutes-1;
GPA-16+RIC-8, 0.35 (0.26-0.43) minutes-1; GOA-1, 0.052
(0.044-0.061) minutes-1; GOA-1+RIC-8, 0.12 (0.093-0.14)
minutes-1. (G,H) Co-immunoprecipitation of the same wild-type
embryonic extracts with GPA-16 (G) or GOA-1 (H) antibodies either alone (lanes
1) or in the presence of 100 µM GDP (lanes 2) or 100 µM GTP-S
(lanes 3). The co-immunoprecipitated material was detected using RIC-8,
GPR-1/2, GPA-16 or GOA-1 antibodies, as indicated on the left with arrowheads.
A previous study reported that spindle positioning in ric-8(md1909)
goa-1(RNAi) embryos is similar to that of ric-8(md1909) embryos
(Couwembergs et al., 2004); the
difference with our results may reflect the use of distinct RNAi
conditions.
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