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 "dotblot" 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-␦ 1 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)P 3 ) binding) binds specifically to D-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P 4 ) (the PtdIns(3,4,5)P 3 headgroup) with 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 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)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 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.

SUMMARY Transmembrane signaling by the epidermal growth factor receptor (EGFR) involves ligand-induced dimerization and allosteric regulation of the intracellular tyrosine kinase domain. Crystallographic studies have shown how ligand binding induces dimerization of the EGFR extracellular region, but cannot explain the ‘high-affinity’ and ‘low-affinity’ classes of cell-surface EGF-binding sites inferred from curved Scatchard plots. From a series of crystal structures of the Drosophila EGFR extracellular region, we show here how Scatchard plot curvature arises from negatively cooperative ligand binding. The first ligand-binding event induces formation of an asymmetric dimer with only one bound ligand. The unoccupied site in this dimer is structurally restrained, leading to reduced affinity for binding of the second ligand, and thus negative cooperativity. Our results explain the cell-surface binding characteristics of EGF receptors, and suggest how individual EGFR ligands might stabilize distinct dimeric species with different signaling properties.

The dynamins are 100-kDa GTPases involved in the scission event required for formation of endocytotic vesicles. The two main described mammalian dynamins (dynamin؊1 and dynamin؊2) both contain a pleckstrin homology (PH) domain, which has been implicated in dynamin binding to (and activation by) acidic phospholipids, most notably phosphoinositides. We demonstrate that the PH domains of both dynamin isoforms require oligomerization for high affinity phosphoinositide binding. Strong phosphoinositide binding was detected only when the PH domains were dimerized by fusion to glutathione S-transferase, or via a single engineered intermolecular disulfide bond. Phosphoinositide binding specificities agreed reasonably with reported effects of different phospholipids on dynamin GTPase activity. Although they differ in their ability to inhibit rapid endocytosis in adrenal chromaffin cells, the dynamin؊1 and dynamin؊2 PH domains showed identical phosphoinositide binding specificities. Since oligomerization is required for binding of the dynamin PH domain to phosphoinositides, it follows that PH domain-mediated phosphoinositide binding will favor oligomerization of intact dynamin (which has an inherent tendency to self-associate). We propose that the dynamin PH domain thus mediates the observed cooperative binding of dynamin to membranes containing acidic phospholipids and promotes the self-assembly that is critical for both stimulation of its GTPase activity and its ability to achieve membrane scission.

Myeloproliferative neoplasms (MPNs) are blood cancers characterized by excessive production of mature myeloid cells, which result from the acquisition of somatic driver mutations in hematopoietic stem cells (HSCs). Epidemiologic studies indicate a substantial disease heritability that is among the highest known for cancers 1 . However, only a limited set of genetic risk loci have been identified, and the underlying biological mechanisms leading to MPN acquisition remain unexplained. Here, we conducted a large-scale genome-wide association study (3,797 cases and 1,152,977 controls) to identify 17 MPN risk loci (p < 5.0 × 10 −8 ), seven of which have not been previously reported. We find a shared genetic architecture between MPN risk and several hematopoietic traits spanning distinct lineages, an enrichment for risk variants mapping to accessible chromatin in HSCs, and associations of increased MPN risk with longer leukocyte telomere length and other clonal hematopoietic states, collectively implicating HSC function and self-renewal. Gene mapping identifies modulators of HSC biology and targeted variant-to-function assays suggest likely roles for CHEK2 and GFI1B in altering HSC function to confer disease risk. Overall, we demonstrate the power of human genetic studies to illuminate a previously unappreciated mechanism for inherited MPN risk through modulation of HSC function.