Pleckstrin homology (PH) domains are small protein modules of around 120 amino acids found in many proteins involved in cell signalling, cytoskeletal rearrangement and other processes. Although several different protein ligands have been proposed for PH domains, their only clearly demonstrated physiological function to date is to bind membrane phosphoinositides. The PH domain from phospholipase C-delta(1) binds specifically to PtdIns(4,5)P(2) and its headgroup, and has become a valuable tool for studying cellular PtdIns(4,5)P(2) functions. More recent developments have demonstrated that a subset of PH domains recognizes the products of agonist-stimulated phosphoinositide 3-kinases. Fusion of these PH domains to green fluorescent protein has allowed dramatic demonstrations of their independent ability to drive signal-dependent recruitment of their host proteins to the plasma membrane. We discuss the structural basis for this 3-phosphoinoistide recognition and the role that it plays in cellular signalling. PH domains that bind specifically to phosphoinositides comprise only a minority (perhaps 15%) of those known, raising questions as to the physiological role of the remaining 85% of PH domains. Most (if not all) PH domains bind weakly and non-specifically to phosphoinositides. Studies of dynamin-1 have indicated that oligomerization of its PH domain may be important in driving membrane association. We discuss the possibility that membrane targeting by PH domains with low affinity for phosphoinositides could be driven by alteration of their oligomeric state and thus the avidity of their membrane binding.

We have examined the differential binding of Hck and Fyn to HIV‐1 Nef to elucidate the structural basis of SH3 binding affinity and specificity. Full‐length Nef bound to Hck SH3 with the highest affinity reported for an SH3‐mediated interaction (KD 250 nM). In contrast to Hck, affinity of the highly homologous Fyn SH3 for Nef was too weak (KD > 20 microM) to be accurately determined. We show that this distinct specificity lies in a variable loop, the ‘RT loop’, positioned close to conserved SH3 residues implicated in the binding of proline‐rich (PxxP) motifs. A mutant Fyn SH3 with a single amino acid substitution (R96I) in its RT loop had an affinity (KD 380 nM) for Nef comparable with that of Hck SH3. Based on additional mutagenesis studies we propose that the selective recognition of Nef by Hck SH3 is determined by hydrophobic interactions involving an isoleucine residue in its RT loop. Although Nef contains a PxxP motif which is necessary for the interaction with Hck SH3, high affinity binding was only observed for intact Nef protein. The binding of a peptide containing the Nef PxxP motif showed > 300‐fold weaker affinity for Hck SH3 than full‐length Nef.

Pleckstrin homology (PH) domains represent the 11 th most common domain in the human proteome. They are best known for their ability to bind phosphoinositides with high affinity and specificity, although it is now clear that less than 10% of all PH domains share this property. Cases in which PH domains bind specific phosphoinositides with high affinity are restricted to those phosphoinositides that have a pair of adjacent phosphates in their inositol headgroup. Those that do not (PtdIns3P, PtdIns5P and PtdIns(3,5)P 2 ) are instead recognized by distinct classes of domains including FYVE domains, phox homology (PX) domains, PHD fingers, and the recently identified PROPPINs. Of the 90% of PH domains that do not bind strongly and specifically to phosphoinositides, few are well understood. One group of PH domains appears to bind both phosphoinositides (with little specificity) and Arf family small G-proteins, and are targeted to the Golgi apparatus where both phosphoinositides and the relevant Arfs are both present. Here, the PH domains may function as coincidence detectors. A central challenge in understanding the majority of PH domains to establish whether the very low affinity phosphoinositide binding reported in many cases has any functional relevance. For PH domains from dynamin and from Dbl family proteins, this weak binding does appear to be functionally important, although its precise mechanistic role is unclear. In many other cases, it is quite likely that alternative binding partners are more relevant, and that the observed PH domain homology represents conservation of structural fold rather than function.

PH domains (pleckstrin homology domains) are the 11th most common domain in the human genome and are best known for their ability to target cellular membranes by binding specifically to phosphoinositides. Recent studies in yeast have shown that, in fact, this is a property of only a small fraction of the known PH domains. Most PH domains are not capable of independent membrane targeting, and those capable of doing so (approx. 33%) appear, most often, to require both phosphoinositide and non-phosphoinositide determinants for their subcellular localization. Several recent studies have suggested that small GTPases such as ARF family proteins play a role in defining PH domain localization. Some others have described a signalling role for PH domains in regulating small GTPases, although phosphoinositides may also play a role. These findings herald a change in our perspective of PH domain function, which will be significantly more diverse than previously supposed.