Response to Chabre et al.

In their letter, Chabre, Antonny and Paris state that pleckstrin homology (PH) domains are the sole arbiter of phosphatidylinositol (4,5)-bisphosphate (PIP2) interactions with proteins. This is misleading for two reasons: (1) PH domains are found on a diverse array of proteins with a wide range of affinities for different phospholipids; (2) it would be surprising if there were only one structural `motif' capable of specific, biologically relevant interactions with any lipid.Several recent reviews (e.g. Ref. [1xLemmon, M.A. et al. Trends Cell Biol. 1997; 7: 237–242Abstract | Full Text PDF | PubMed | Scopus (136)See all References[1]) summarize the current state of our understanding of the roles played by PH domains in both protein and lipid interactions. Structural studies of four PH domains from different proteins reveal a conserved folded structure of orthogonal β sheets with a C-terminal α helix and a positively charged face, comprising three variable loops, which is thought to contact the negatively charged lipid head groups. There is very limited sequence identity amongst the >90 proteins containing PH domains, and thus these positively charged surfaces are highly divergent in structure and binding constants. With the identification of a `positive patch' on one surface of the crystallized ARF1–GDP protein[2xAmor, J.C. et al. Nature. 1994; 372: 704–708Crossref | PubMedSee all References[2]and the long history of phospholipid dependence on ARF activation and membrane association, it is reasonable to speculate that this patch on ARF is the site of PIP2 binding. Mutagenesis studies have since confirmed the importance of these conserved lysine and arginine residues in ARF–PIP2 interactions.Can I prove that this lipid binding site is biologically relevant? No, but neither can one do so for PH domains. Indeed, there is evidence that, in a few instances, the interactions of PH domains with substances other than PIP2 must be involved in membrane associations[1xLemmon, M.A. et al. Trends Cell Biol. 1997; 7: 237–242Abstract | Full Text PDF | PubMed | Scopus (136)See all References[1].Can I prove that PIP2 is the biologically important ligand on ARFs? No, and neither should one conclude that it is for PH domains. With higher phosphorylated forms of phosphatidylinositol still being discovered, it seems foolish to limit any model of ARF or PH domain binding to PIP2.With regard to the methodological differences leading to different conclusions, I would like to recommend a very nice study by Zheng et al.[3xZheng, C.J. et al. J. Mol. Biol. 1996; 255: 14–21Crossref | PubMed | Scopus (194)See all References[3]. These authors report the failure to observe binding of the PH domain of dynamin to PIP2 in lipid bilayers or in gel filtration-based assay, as was observed for other PH domains. However, using detergent-solubilized lipids in a fluorescence-quenching assay, they report the binding of PI(4)P and PIP2 with apparent Kds of 1.8 and 4.4 μm (pleckstrin and ARF have apparent binding constants for PIP2 of 30 and 45 μm, respectively). Zheng et al. conclude that `the physical state of the phospholipids recognized by the dynamin PH domain therefore appears to be different from that for other PH domains'[3xZheng, C.J. et al. J. Mol. Biol. 1996; 255: 14–21Crossref | PubMed | Scopus (194)See all References[3]. Should we then conclude that the binding of pleckstrin or dynamin to PIP2 is artefactual?Finally, the key observations in the paper by Terui et al.[4xTerui, T. et al. J. Biol. Chem. 1994; 269: 28130–28135PubMedSee all References[4]are misrepresented by Chabre and co-workers. The two experiments reported in Ref. [4xTerui, T. et al. J. Biol. Chem. 1994; 269: 28130–28135PubMedSee all References[4]and discussed in Chabre's letter were measuring different processes. In the first (see Fig. 2 in Ref. [4xTerui, T. et al. J. Biol. Chem. 1994; 269: 28130–28135PubMedSee all References[4]), we documented the effects of PIP2 and other lipids on the rate of dissociation of GDP from recombinant ARF1. The rate of release of pre-bound [3H]GDP was monitored in excess unlabeled GDP, to prevent re-association of the label. Thus, this result documents the dramatic effect of PIP2 on GDP release, but gives no clue as to whether or not the nucleotide binding site is occupied with unlabeled GDP. Figure 3 in Ref. [4xTerui, T. et al. J. Biol. Chem. 1994; 269: 28130–28135PubMedSee all References[4]shows the lack of effect of PIP2 on release of GTPγS, and clearly shows no `denaturing' effect of the PIP2 on the ARF because the GTPγS binding is unaffected at times and under conditions in which the PIP2 is active on ARF–GDP. We then went on to show that, in the absence of added nucleotides, the nucleotide-free ARF1 is stable at 30°C for over an hour in the presence of PIP2. This is in marked contrast to nucleotide-free ARF1, obtained by renaturation of ARF1 out of 8 m urea, which binds guanine nucleotides very rapidly but fails to do so after an incubation of even 15 min at 30°C without nucleotides. Figure 5 in Ref. [4xTerui, T. et al. J. Biol. Chem. 1994; 269: 28130–28135PubMedSee all References[4]goes on to show that dilution of the PIP2 results in a six-fold enhancement of GTPγS binding to ARF1 over the same protein incubated without PIP2. How Chabre and co-workers interpret this as `a progressive and complete loss of the capacity the empty ARF had initially to bind GTP' is mysterious. Equally mysterious to me is the last sentence in their letter, which suggests that a nucleotide-free ARF protein is not an intermediate in the activation of that protein.I am sure that a careful reading of the published work on PH domains, ARF, Cdc42 and other phosphatidylinositol polyphosphate binding proteins will reveal many interesting and unresolved issues that will form the basis for future experimentation. It is only through the development of better tools and cell assay systems that we can begin to unravel the complexities of protein–lipid interactions, which are currently only poorly being mimicked in the test tube.