The functional relevance of low affinity phosphoinositide binding by the PH domain in Dbl family guanine nucleotide exchange factors

Low affinity phosphoinositide binding is found in several phosphoinositide recognition domains, including PH domains, and members of the ESCRT complex. In light of the numerous occurrences of these domains in the human proteome, the significance of low affinity phosphoinositide binding has become an important question. We have investigated this question in the PH domain present in Dbl family guanine nucleotide exchange factors (GEFs) and in the ESCRT proteins. We found that while the PH domain in several Dbl family members bound phosphoinositides with low affinity, this property of the PH domain appeared critical for the in vivo GEF activity of the adjacent DH domain. The Dbs DH/PH fragment bound PtdIns(4,5)P2 in vitro with low affinity (∼10 muM), and localized to the cytosol when expressed in vivo as a GFP fusion. However, forced dimerization led to plasma membrane recruitment of the DH/PH fragment, an effect that required the phosphoinositide binding properties of the PH domain. Constitutive plasma membrane targeting of this fragment by a fused PLC-gamma1 PH domain or farnesylation signal, significantly enhanced in vivo GEF activity toward Cdc42 and RhoA, suggesting the PH domain may regulate GEF activity through a cooperative membrane recruiting mechanism. Interestingly, PH domain mutations within a membrane targeted DH/PH fragment led to a significant reduction in Cdc42 and RhoA activation, suggesting an additional regulatory role by the PH domain, such as an allosteric mechanism. In contrast, the Tiam-1 GST-DH/PH fragment bound specifically, though weakly, to PtdIns-3-P in vitro but did not target, when dimerized, to identifiable cellular membranes in vivo. PH domain mutations that abolished PtdIns-3-P binding did not alter bulk localization of Tiam-1, but dramatically impaired in vivo GEF activity, suggesting an non-recruiting regulatory role for the Tiam-1 PH domain, such as an allosteric one. Finally, we investigated the phosphoinositide binding properties of mVps24p and the yeast ESCRT proteins, and found that most bound phosphoinositides promiscuously and in the case of mVps24p, with low affinity, suggesting ESCRT proteins may be recruited to late endosome membranes, by a cooperative mechanism involving weak non-specific phosphoinositide interactions combined with other determinants, such as specific association with ubiquitin.