IDENTIFICATION OF THE BINDING-SITE FOR ACIDIC PHOSPHOLIPIDS ON THE PHDOMAIN OF DYNAMIN - IMPLICATIONS FOR STIMULATION OF GTPASE ACTIVITY

It has recently been suggested that pleckstrin homology (PH) domains b ind specifically to phospholipids, with phosphatidylinositol-4,5-bisph osphate (PtdIns(4,5)P-z) being most strongly bound. This observation s uggests that PH domains may be responsible for membrane association of proteins in which they occur. Further, this membrane association may be regulated by enzymes that modify lipid head groups to which PH doma ins may bind. We have studied the binding of phospholipids to the PH d omain of human dynamin, a 100 kDa GTPase that is involved in the initi al stages of endocytosis. We describe a rapid method for screening PH domain/ligand interactions that gives precise binding constants. We co nfirm that PtdIns(4,5)P-2 can bind to dynamin PH domain, although not in an aggregated state. Using NMR spectroscopy, we have mapped a speci fic site on the surface of dynamin PH domain of which binding of gIns( 1,4,5)P-3 (the head-group skeleton of PtdIns(4,5)P-2) occurs. The rela tive affinity of acidic phospholipids for dynamin PH domain correlates with their ability to activate the GTPase of dynamin. We propose, the refore, that the interaction of these phospholipids with dynamin is li kely to occur aia the PH domain. Given the fact that PH domains are of ten found in proteins associated with GTPase activity, or in guanine n ucleotide exchange factors, we suggest that one role of PH domains may be to couple phosphatidylinositol signalling to GTP hydrolysis. (C) 1 996 Academic Press Limited