ABSTRACT
Dynamin is required for clathrin-mediated endocytosis (CME). Its GTPase activity is stimulated by phospholipid binding to its PH domain, which induces helical oligomerization. We have designed a series of novel pyrimidine-based "Pyrimidyn" compounds that inhibit the lipid-stimulated GTPase activity of full length dynamin I and II with similar potency. The most potent analogue, Pyrimidyn 7, has an IC50 of 1.1 µM for dynamin I and 1.8 µM for dynamin II, making it among the most potent dynamin inhibitors identified to date. We investigated the mechanism of action of the Pyrimidyn compounds in detail by examining the kinetics of Pyrimidyn 7 inhibition of dynamin. The compound competitively inhibits both GTP and phospholipid interactions with dynamin I. While both mechanisms of action have been previously observed separately, this is the first inhibitor series to incorporate both and thereby to target two distinct domains of dynamin. Pyrimidyn 6 and 7 reversibly inhibit CME of both transferrin and EGF in a number of non-neuronal cell lines as well as inhibiting synaptic vesicle endocytosis (SVE) in nerve terminals. Therefore, Pyrimidyn compounds block endocytosis by directly competing with GTP and lipid binding to dynamin, limiting both the recruitment of dynamin to membranes and its activation. This dual mode of action provides an important new tool for molecular dissection of dynamin's role in endocytosis.
Subject(s)
Drug Design , Dynamins/antagonists & inhibitors , Pyrimidines/chemistry , Pyrimidines/chemical synthesis , Small Molecule Libraries/chemical synthesis , Animals , Biological Assay , Blotting, Western , COS Cells , Chlorocebus aethiops , Endocytosis/drug effects , Flow Cytometry , Molecular Structure , Protein Binding/drug effects , Pyrimidines/pharmacology , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacologyABSTRACT
Depolarization of nerve terminals stimulates rapid dephosphorylation of two isoforms of dynamin I (dynI), mediated by the calcium-dependent phosphatase calcineurin (CaN). Dephosphorylation at the major phosphorylation sites Ser-774/778 promotes a dynI-syndapin I interaction for a specific mode of synaptic vesicle endocytosis called activity-dependent bulk endocytosis (ADBE). DynI has two main splice variants at its extreme C terminus, long or short (dynIxa and dynIxb) varying only by 20 (xa) or 7 (xb) residues. Recombinant GST fusion proteins of dynIxa and dynIxb proline-rich domains (PRDs) were used to pull down interacting proteins from rat brain nerve terminals. Both bound equally to syndapin, but dynIxb PRD exclusively bound to the catalytic subunit of CaNA, which recruited CaNB. Binding of CaN was increased in the presence of calcium and was accompanied by further recruitment of calmodulin. Point mutations showed that the entire C terminus of dynIxb is a CaN docking site related to a conserved CaN docking motif (PXIXI(T/S)). This sequence is unique to dynIxb among all other dynamin variants or genes. Peptide mimetics of the dynIxb tail blocked CaN binding in vitro and selectively inhibited depolarization-evoked dynI dephosphorylation in nerve terminals but not of other dephosphins. Therefore, docking to dynIxb is required for the regulation of both dynI splice variants, yet it does not regulate the phosphorylation cycle of other dephosphins. The peptide blocked ADBE, but not clathrin-mediated endocytosis of synaptic vesicles. Our results indicate that Ca(2+) influx regulates assembly of a fully active CaN-calmodulin complex selectively on the tail of dynIxb and that the complex is recruited to sites of ADBE in nerve terminals.
