Specific and high-affinity binding of inositol phosphates to an isolated pleckstrin homology domain.

Abstract

Pleckstrin homology (PH) domains are found in many signaling molecules and are thought to be involved in specific intermolecular interactions. Their binding to several proteins and to membranes containing 1-Caphosphatidylinositol 4,5-bisphosphate [PtdIns(4,5) The importance of modular binding domains in regulating interactions between signaling modules, as well as their activity, is well established (1, 2). The pleckstrin homology (PH) domain is thought to represent such a module. It contains '120 aa and was identified.as a region of sequence homology with pleckstrin that appears in a large variety of proteins involved in intracellular signaling (3)(4)(5)(6)(7)(8). Recent x-ray crystallography (9, 10) and NMR (11-14) studies have demonstrated that PH domains have a distinct characteristic structure. Their fold is best described as a seven-stranded ,-sandwich of two orthogonal 1-sheets, closed off at one corner by a C-terminal a-helix. The domain is electrostatically polarized, with the three most variable loops falling in the most positively charged surface (9).Reported ligands for PH domains include 13y subunits (Gg,) of heterotrimeric G proteins (15,16) We report here that the isolated PH domain of PLC-81 interacts specifically, and with high affinity, with both Ptdlns(4,5)P2 and Ins(1,4,5)P3. The PH domain is, therefore, likely to represent the portion of PLC-&1 responsible for high-affinity binding to PtdIns(4,5)P2 and for the observed product inhibition by Ins(1,4,5)P3. These results are a demonstration of a stereo-specific high-affinity ligand for a PH domain and support a proposed role for PH domains in regulation of PLC isoforms. MATERIALS AND METHODSGeneration of Recombinant PH Domains. Dynamin PH (DynPH) was produced in Escherichia coli as described (9). Constructs expressing residues 11-140 of rat PLC-81 (28) (PLCS-PH) and residues 1-105 of pleckstrin (29) (PlecN-PH) were produced and purified as described (9,30

Determining selectivity of phosphoinositide-binding domains

“…However, when PH or FYVE domain binding to phosphoinositides is studied, there appears to be more of a discrepancy. Monitoring steady state RU signals, we achieve excellent agreement in K D values (0.6-3 µM) for PtdIns(4,5)P 2 binding by the PLC 1 PH domain between our SPR studies [56,57], ITC [39], and centrifugation approaches [20]. Similarly, estimates for PtdIns3P binding by the Hrs1 FYVE domain are within 2-fold when comparing results from SPR (K D = 4.8 µM [57]) and centrifugation assays (K D = 2.5 µM [46]).…”

“…There was some confusion in the literature as to whether the isolated dynamin PH domain binds phosphoinositides. Our own dot-blot approaches suggested that dynamin's PH domain binds all phosphoinositides (see Figure 1), but our attempts to quantitate its binding to vesicles containing PtdIns(4,5)P 2 had failed [39], suggesting that the interaction may be very weak. NMR-based K D measurements for binding of the PH domain to isolated PtdIns(4,5)P 2 headgroup were in the millimolar range [47,48], consistent with this observation.…”

“…We previously employed isothermal titration calorimetry (ITC) to assess binding of the PLC 1 PH domain to artificial membranes containing PtdIns(4,5)P 2 [39]. Limit-sonicated dimyristoylphosphatidylcholine (DMPC) vesicles containing 5% (mole/mole) dipalmitoyl PtdIns(4,5)P 2 were placed in the calorimeter cell, and aliquots of purified PLC 1 PH domain (at 120 µM) were injected during the titration.…”

Sprouty4 negatively regulates protein kinase C activation by inhibiting phosphatidylinositol 4,5-biphosphate hydrolysis

“…GFP-PLCd-1 PH domain selectively binds to both PIP 2 and IP 3 , and PLC activation triggered dissociation of the PH domain of PLCd-1 from the plasma membrane to cytosol, which represents PIP 2 hydrolysis and IP 3 production in the cytosol (Lemmon et al, 1995). In our study of live cell imaging, VEGF-A induced rapid translocation of GFP-PH domain from plasma membrane to cytosol in 293T-VEGFR-2 cells, whereas Sprouty4 overexpression strongly inhibited mobilization of GFP-PH domain (Figure 4c).…”

“…To assess the stimuli-induced PIP 2 hydrolysis, we used GFP-tagged PH domain of PLCd-1 (received from Professor T Sasaki, Department of Pathology and Immunology, Akita University School of Medicine), which was established as an in vivo fluorescent indicator for PIP 2 (Lemmon et al, 1995). Experimental procedures were the same as the observation of GFP-tagged MARCKS and PKC translocation.…”