The R6A-1 peptide binds to switch II of Galphai1 but is not a GDP-dissociation inhibitor.

Heterotrimeric G-proteins are molecular switches that convert signals from membrane receptors into changes in intracellular physiology. Recently, several peptides that bind heterotrimeric G-protein alpha subunits have been isolated including the novel Galpha(i1).GDP binding peptides R6A and KB-752. The R6A peptide and its minimized derivative R6A-1 interact with Galpha(i1).GDP. Based on spectroscopic analysis of BODIPYFL-GTPgammaS binding to Galpha(i1), it has been reported that R6A-1 has guanine nucleotide dissociation inhibitor (GDI) activity against Galpha(i1) [W.W. Ja, R.W. Roberts, Biochemistry 43 (28) (2004) 9265-9275]. Using radioligand binding, we show that R6A-1 is not a GDI for Galpha(i1) subunits. Furthermore, we demonstrate that R6A-1 reduces the fluorescence quantum yield of the Galpha(i1)-BODIPYFL-GTPgammaS complex, thus explaining the previously reported GDI activity as a fluorescence artifact. We further show that R6A-1 has significant sequence similarity to the guanine nucleotide exchange factor peptide KB-752 that binds to switch II of Galpha(i1). We use competitive binding analysis to show that R6A-1 also binds to switch II of Galpha subunits.