Figure 5.

DAPLE inhibits nucleotide exchange on Gα_s via its GBA motif. A and B, DAPLE GBA peptide decreases the steady-state GTPase activity of Gα_s. A representative time course of the steady-state GTPase activity of His–Gα_s alone (black), in the presence of DAPLE GBA peptide (30 μm, red), or control peptide (30 μm, blue) is shown in A, and quantification of the dose-dependent effect of the peptides is shown in B (mean ± S.E., n = 3). Results are presented as raw production of free [³²P]P_i (pmol) in A or percent change relative to the production of free [³²P]P_i by Gα_s alone at 10 min (% of control) in B. Average IC₅₀ value was determined as described under “Experimental procedures.” C and D, DAPLE GBA peptide decreases the rate of GTPγS binding to Gα_s. A representative time course of [³⁵S]GTPγS binding to His–Gα_s in the absence (black) or presence of DAPLE GBA peptide (30 μm, red) is shown in C, and quantification of the dose-dependent effect of the DAPLE GBA peptide is shown in D (mean ± S.E., n = 3). Results are presented as raw [³⁵S]GTPγS binding (picomoles) in C or percent change relative to [³⁵S]GTPγS binding to Gα_s alone at 10 min (% of control) in D. Rate constants and average IC₅₀ values were determined as described under “Experimental procedures,” E and F, purified DAPLE WT (amino acids 1650–2028), but not F1675A mutant, decreases GTPγS binding to Gα_s. A representative time course of [³⁵S]GTPγS binding to His–Gα_s in the absence (black) or presence of purified His–DAPLE (9 μm, red) is shown in E, and quantification of the effect of His–DAPLE WT (3.3 μm, red) compared with His–DAPLE F1675A (3.3 μm, blue) is shown in D (mean ± S.E., n = 3). Results are presented as raw [³⁵S]GTPγS binding (picomoles) in E or percent change relative to [³⁵S]GTPγS binding to Gα_s alone at 10 min (% of control) in F. Rate constants determined as described under “Experimental procedures.”