Interaction in vitro; bait produced as a recombinant fusion protein in bacterial system; prey derived from wild-type adult extract.

Gαo bound with GDP compleses more effectivvely with pins than Gαo bound with GTP.

Two-hybrid system: yeast LexA-BD/GAL4-AD

72 pins clones recovered in the Gαo screen.

Source was yeast cell line; bait produced as transgenic fusion protein; prey produced as transgenic fusion protein (prey from adult head cDNA expression library).

Phosphorylated pins from adult head extract binds Gαo only in the presence of GDP, not GTP-γS.

Interaction in vitro; bait produced as a recombinant fusion protein in bacterial system; prey derived from wild-type adult head extract.

Interaction in vitro; bait produced as a recombinant fusion protein in bacterial system; prey derived from wild-type adult head extract.

The pins GoLoco domain1 binds both GDP- and GTP-bound forms of Gαo, while pins GoLoco domain 3 binds only the GDP-bound form of Gαo.

raps protein interacts with wildtype, constitutively GDP-bound (G203T) and constitutively GTP-bound (Q205L) forms of G-oα47A.

raps protein interacts more strongly with a GTP-bound (Q205L) mutant of G-oα47A than with a GDP-bound mutant (G203T).

G-oα47A and G-iα65A compete for binding of raps protein.

Bait expressed in transiently transfected cells.

Prey expressed in transiently transfected cells.

This interaction is abolished in Tre1 mutants.

The requirement for Tre1 in vivo implies that activation of G-oα47A is required for raps binding, contradicting in vitro data indicating that raps binds both GTP- and GDP-bound forms of G-oα47A. This could reflect competitive binding in vivo.

The dansyl group acts as an acceptor of excited tryptophan residues in FRET experiments.

Interaction in vitro; fluorescence donor produced as a recombinant fusion protein in bacterial system; fluorescence acceptor peptide synthesized and labeled in vitro.