SPECIFICITY AND PROMISCUITY IN PHOSPHOINOSITIDE BINDING BY PLECKSTRINHOMOLOGY DOMAINS

Pleckstrin homology (PH) domains are small protein modules involved in recruitment of signaling molecules to cellular membranes, in some cas es by binding specific phosphoinositides. We describe use of a conveni ent ''dot-blot''' approach to screen 10 different PH domains for those that recognize particular phosphoinositides. Each PH domain bound pho sphoinositides in the assay, but only two (from phospholipase C-delta( 1) and Grp1) showed clear specificity for a single species. Using solu ble inositol phosphates, we show that the Grp1 PH domain (originally c loned on the basis of its phosphatidylinositol 3,4,5-trisphosphate (Pt dIns(3,4,5)P-3) binding) binds specifically to D-myo-inositol 1,3,4,5- tetrakisphosphate (Ins(1,3,4,5)P-4) (the PtdTns(3,4,5)P-3 headgroup) w ith K-D = 27.3 nM, but binds D-myo-inositol 1,3,4-trisphosphate (Ins(1 ,3,4)P-3) or D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P-3) over 8 0-fold more weakly. We show that this specificity allows localization of the Grp1 PH domain to the plasma membrane of mammalian cells only w hen phosphatidylinositol 3-kinase (PI 3-K) is activated. The presence of three adjacent equatorial phosphate groups was critical for inosito l phosphate binding by the Grp1 PH domain. By contrast, another PH dom ain capable of PI 3-K-dependent membrane recruitment (encoded by EST68 4797) does not distinguish Ins(1,3,4)P-3 from Ins(1,3,4,5)P-3 (binding both with very high affinity), despite selecting strongly against Ins (1,4,5)P-3. The remaining PH domains tested appear significantly less specific for particular phosphoinositides. Together with data presente d in the literature, our results suggest that many PH domains bind sim ilarly to multiple phosphoinositides (and in some cases phosphatidylse rine), and are likely to be regulated in vivo by the most abundant spe cies to which they bind. Thus, using the same simple approach to study several PH domains simultaneously, our studies suggest that highly sp ecific phosphoinositide binding is a characteristic of relatively few cases.