title: Identification of the binding site for acidic phospholipids on the pH domain of dynamin: implications for stimulation of GTPase activity.
abstract: It has recently been suggested that pleckstrin homology (PH) domains bind specifically to phospholipids, with phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) being most strongly bound. This observation suggests 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 domains may bind. We have studied the binding of phospholipids to the PH domain of human dynamin, a 100 kDa GTPase that is involved in the initial stages of endocytosis. We describe a rapid method for screening PH domain/ligand interactions that gives precise binding constants. We confirm that PtdIns(4,5)P2 can bind to dynamin PH domain, although not in an aggregated state. Using NMR spectroscopy, we have mapped a specific site on the surface of dynamin PH domain of which binding of gIns(1,4,5)P3 (the head-group skeleton of PtdIns(4,5)P2) occurs. The relative affinity of acidic phospholipids for dynamin PH domain correlates with their ability to activate the GTPase of dynamin. We propose, therefore, that the interaction of these phospholipids with dynamin is likely to occur via the PH domain. Given the fact that PH domains are often found in proteins associated with GTPase activity, or in guanine nucleotide exchange factors, we suggest that one role of PH domains may be to couple phosphatidylinositol signalling to GTP hydrolysis.
authors.affiliation: Rockefeller University, New York, NY 10021, USA.
authors.forename: J
authors.initials: J
authors.lastname: Zheng
authors.forename: S M
authors.initials: SM
authors.lastname: Cahill
authors.forename: M A
authors.initials: MA
authors.lastname: Lemmon
authors.forename: D
authors.initials: D
authors.lastname: Fushman
authors.forename: J
authors.initials: J
authors.lastname: Schlessinger
authors.forename: D
authors.initials: D
authors.lastname: Cowburn
mesh_terms: D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry*; D034281:Dynamins; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010712:Phosphatidic Acids / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010750:Phosphoproteins; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence
chemical_list: D001798:Blood Proteins; D010712:Phosphatidic Acids; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010750:Phosphoproteins; C039077:platelet protein P47; D020558:GTP Phosphohydrolases; D034281:Dynamins