DAPLE protein inhibits nucleotide exchange on Gαs and Gαq via the same motif that activates Gαi

Abstract

Besides being regulated by G-protein-coupled receptors, the activity of heterotrimeric G proteins is modulated by many cytoplasmic proteins. GIV/Girdin and DAPLE (<u>D</u>vl-<u>a</u>ssociating <u>p</u>rotein with a high frequency of <u>le</u>ucine) are the best-characterized members of a group of cytoplasmic regulators that contain a Gα-binding and -activating (GBA) motif and whose dysregulation underlies human diseases, including cancer and birth defects. GBA motif-containing proteins were originally reported to modulate G proteins by binding Gα subunits of the G_i/o family (Gα_i) over other families (such as G_s, G_q/11, or G_12/13), and promoting nucleotide exchange <i>in vitro</i> However, some evidence suggests that this is not always the case, as phosphorylation of the GBA motif of GIV promotes its binding to Gα_s and inhibits nucleotide exchange. The G-protein specificity of DAPLE and how it might affect nucleotide exchange on G proteins besides Gα_i remain to be investigated. Here, we show that DAPLE's GBA motif, in addition to Gα_i, binds efficiently to members of the G_s and G_q/11 families (Gα_s and Gα_q, respectively), but not of the G_12/13 family (Gα₁₂) in the absence of post-translational phosphorylation. We pinpointed Met-1669 as the residue in the GBA motif of DAPLE that diverges from that in GIV and enables better binding to Gα_s and Gα_q Unlike the nucleotide-exchange acceleration observed for Gα_i, DAPLE inhibited nucleotide exchange on Gα_s and Gα_q These findings indicate that GBA motifs have versatility in their G-protein-modulating effect, <i>i.e.</i> they can bind to Gα subunits of different classes and either stimulate or inhibit nucleotide exchange depending on the G-protein subtype.