Abstract
Pleckstrin homology (PH) domains are small protein modules involved in recruitment of signaling molecules to cellular membranes, in some cases by binding specific phosphoinositides. We describe use of a convenient "dot-blot" approach to screen 10 different PH domains for those that recognize particular phosphoinositides. Each PH domain bound phosphoinositides in the assay, but only two (from phospholipase C-delta1 and Grp1) showed clear specificity for a single species. Using soluble inositol phosphates, we show that the Grp1 PH domain (originally cloned on the basis of its phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) binding) binds specifically to D-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) (the PtdIns(3,4,5)P3 headgroup) with KD = 27.3 nM, but binds D-myo-inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) or D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) over 80-fold more weakly. We show that this specificity allows localization of the Grp1 PH domain to the plasma membrane of mammalian cells only when phosphatidylinositol 3-kinase (PI 3-K) is activated. The presence of three adjacent equatorial phosphate groups was critical for inositol phosphate binding by the Grp1 PH domain. By contrast, another PH domain capable of PI 3-K-dependent membrane recruitment (encoded by EST684797) does not distinguish Ins(1,3,4)P3 from Ins(1,3,4,5)P3 (binding both with very high affinity), despite selecting strongly against Ins(1,4,5)P3. The remaining PH domains tested appear significantly less specific for particular phosphoinositides. Together with data presented in the literature, our results suggest that many PH domains bind similarly to multiple phosphoinositides (and in some cases phosphatidylserine), and are likely to be regulated in vivo by the most abundant species to which they bind. Thus, using the same simple approach to study several PH domains simultaneously, our studies suggest that highly specific phosphoinositide binding is a characteristic of relatively few cases.
Highlights
Pleckstrin homology (PH) domains are small protein modules involved in recruitment of signaling molecules to cellular membranes, in some cases by binding specific phosphoinositides
An Initial Screen Indicates That PH Domains Differ in Both Preferred Ligand and Degree of Specificity—As shown in Fig. 1, the 10 PH domains tested in our initial screen all appeared to bind phosphoinositides. 32P-Labeled GST alone gave no signals above background
For the PLC-␦1 PH domain (PLC␦-PH), we and others have previously shown that the PtdIns(4,5)P2 headgroup (Ins(1,4,5)P3) binds significantly more strongly than any other inositol phosphate (14, 16)
Summary
Surface receptors) to downstream signaling events (17, 18). This has been most well studied for the PH domain of protein kinase B (PKB), known as Akt and RAC (18, 19), and more recently for PLC-␥1 (20), and PDK-1 (21). Ins(1,3,4,5)P4 (the headgroup of PtdIns(3,4,5)P3) binds the Grp PH domain with a KD of 27 nM, in a highly specific interaction that is sufficient for PI 3-K-dependent plasma membrane recruitment of the PH domain in mammalian cells. Another PH domain that is recruited to the membrane by PI 3-K products (encoded by EST684797; Ref. 52) was found to bind Ins(1,3,4,5)P4 with a similar affinity. Unlike the Grp PH domain, this PH domain did not distinguish between Ins(1,3,4,5)P4 and Ins(1,3,4)P3, despite selecting strongly against Ins(1,4,5)P3 By contrast with these examples, most of the PH domains tested appeared quite promiscuous in their phosphoinositide binding according to the dot-blot assay. The PH domain may provide only a general attraction for the membrane surface that cooperates with a specific regulated interaction mediated by other regions of the host molecule
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