Eileen M. Lafer

Mitogen-Activated Protein Kinase Regulates Dopamine Transporter Surface Expression and Dopamine Transport Capacity

The dopamine transporter (DAT) regulates the clearance of dopamine (DA) released into the extracellular space and is an important site on which psychostimulants act to produce their effects. Here, we show that mitogen-activated protein kinase (MAPK) regulates the transport capacity and intracellular trafficking of DAT. Incubation of striatal synaptosomes or epitope-tagged human DAT (hDAT) human embryonic kidney (HEK) 293 cells with the MAPK kinase (MEK) inhibitors 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto) butadiene and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one decreased DA uptake in a concentration- and time-dependent manner. Kinetic studies revealed a decrease in the capacity of transport (Vmax) but no change in Km. Immunoblotting confirmed labeling of p42 and p44 MAPK in untreated striatal synaptosomes and HEK 293 cells, consistent with constitutive MAPK activation, and the inhibitors used decreased MAPK phosphorylation. Biotinylation and confocal imaging studies showed that MAPK inhibition promoted the clathrin-associated redistribution of hDAT from the plasma membrane to the cytosol. In contrast, transient transfection of hDAT-expressing cells with constitutively active MEK increased the Vmax of DA transport without altering Km. However, only a small increase in hDAT cell surface expression was seen. These data demonstrate an involvement of the MAPK cascade in regulating DAT transport capacity in striatum and that inhibition of this cascade decreases DAT cell surface expression in HEK 293 cells. Furthermore, they highlight the potential role of MAPK as a presynaptic mechanism that regulates DA signaling.

Negatively supercoiled plasmids contain left-handed Z-DNA segments as detected by specific antibody binding

Negative superhelical coiling of covalently closed DNA plasmids facilitates the formation of left-handed Z-DNA segments. This was demonstrated by binding of antibodies specific for Z-DNA. Plasmid pBR322 and two derivatives from it, pLP32 and pLP014, carrying inserts of alternating CG sequences of 32 bp and 14 bp, respectively, were used. Longer inserts required less negative superhelical density to induce the B-Z transitions. Antibody binding to supercoiled plasmids was also visualized by electron microscopy. Cross-linking of the antibody to the negatively supercoiled plasmid and restriction of the DNA with restriction endonucleases demonstrated that the antibodies combine with the CG insert in pLP32. For pBR322, however, evidence suggests that the antibody combines with a section of DNA containing 14 bases with alternating purine and pyrimidine residues with one residue out of alternation: CACGGGTGCGCATG. These cross-linking studies provide evidence for the binding specificity of the anti-Z-DNA antibodies. On the basis of experimental findings, we have calculated the changes in free energy associated with B-Z transitions in superhelical plasmids.

PI 3-kinase regulation of dopamine uptake

The magnitude and duration of dopamine (DA) signaling is defined by the amount of vesicular release, DA receptor sensitivity, and the efficiency of DA clearance, which is largely determined by the DA transporter (DAT). DAT uptake capacity is determined by the number of functional transporters on the cell surface as well as by their turnover rate. Here we show that inhibition of phosphatidylinositol (PI) 3‐kinase with LY294002 induces internalization of the human DAT (hDAT), thereby reducing transport capacity. Acute treatment with LY294002 reduced the maximal rate of [3H]DA uptake in rat striatal synaptosomes and in human embryonic kidney (HEK) 293 cells stably expressing the hDAT (hDAT cells). In addition, LY294002 caused a significant redistribution of the hDAT from the plasma membrane to the cytosol. Conversely, insulin, which activates PI 3‐kinase, increased [3H]DA uptake and blocked the amphetamine‐induced hDAT intracellular accumulation, as did transient expression of constitutively active PI 3‐kinase. The LY294002‐induced reduction in [3H]DA uptake and hDAT cell surface expression was inhibited by expression of a dominant negative mutant of dynamin I, indicating that dynamin‐dependent trafficking can modulate transport capacity. These data implicate DAT trafficking in the hormonal regulation of dopaminergic signaling, and suggest that a state of chronic hypoinsulinemia, such as in diabetes, may alter synaptic DA signaling by reducing the available cell surface DATs.

Structure of the Hsp110:Hsc70 Nucleotide Exchange Machine

Hsp70s mediate protein folding, translocation, and macromolecular complex remodeling reactions. Their activities are regulated by proteins that exchange ADP for ATP from the nucleotide-binding domain (NBD) of the Hsp70. These nucleotide exchange factors (NEFs) include the Hsp110s, which are themselves members of the Hsp70 family. We report the structure of an Hsp110:Hsc70 nucleotide exchange complex. The complex is characterized by extensive protein:protein interactions and symmetric bridging interactions between the nucleotides bound in each partner proteins NBD. An electropositive pore allows nucleotides to enter and exit the complex. The role of nucleotides in complex formation and dissociation, and the effects of the protein:protein interactions on nucleotide exchange, can be understood in terms of the coupled effects of the nucleotides and protein:protein interactions on the open-closed isomerization of the NBDs. The symmetrical interactions in the complex may model other Hsp70 family heterodimers in which two Hsp70s reciprocally act as NEFs.

Mechanical stress-activated integrin α5β1 induces opening of connexin 43 hemichannels

The connexin 43 (Cx43) hemichannel (HC) in the mechanosensory osteocytes is a major portal for the release of factors responsible for the anabolic effects of mechanical loading on bone formation and remodeling. However, little is known about how the Cx43 molecule responds to mechanical stimulation leading to the opening of the HC. Here, we demonstrate that integrin α5β1 interacts directly with Cx43 and that this interaction is required for mechanical stimulation-induced opening of the Cx43 HC. Direct mechanical perturbation via magnetic beads or conformational activation of integrin α5β1 leads to the opening of the Cx43 HC, and this role of the integrin is independent of its association with an extracellular fibronectin substrate. PI3K signaling is responsible for the shear stress-induced conformational activation of integrin α5β1 leading to the opening of the HC. These results identify an unconventional function of integrin that acts as a mechanical tether to induce opening of the HC and provide a mechanism connecting the effect of mechanical forces directly to anabolic function of the bone.

AP180 and AP-2 Interact Directly in a Complex That Cooperatively Assembles Clathrin

Clathrin-coated vesicles are involved in protein and lipid trafficking between intracellular compartments in eukaryotic cells. AP-2 and AP180 are the resident coat proteins of clathrin-coated vesicles in nerve terminals, and interactions between these proteins could be important in vesicle dynamics. AP180 and AP-2 each assemble clathrin efficiently under acidic conditions, but neither protein will assemble clathrin efficiently at physiological pH. We find that there is a direct, clathrin-independent interaction between AP180 and AP-2 and that the AP180-AP-2 complex is more efficient at assembling clathrin under physiological conditions than is either protein alone. AP180 is phosphorylated in vivo, and in crude vesicle extracts its phosphorylation is enhanced by stimulation of casein kinase II, which is known to be present in coated vesicles. We find that recombinant AP180 is a substrate for casein kinase II in vitro and that its phosphorylation weakens both the binding of AP-2 by AP180 and the cooperative clathrin assembly activity of these proteins. We have localized the binding site for AP-2 to amino acids 623–680 of AP180. The AP180/AP-2 interaction can be disrupted by a recombinant AP180 fragment containing the AP-2 binding site, and this fragment also disrupts the cooperative clathrin assembly activity of the AP180-AP-2 complex. These results indicate that AP180 and AP-2 interact directly to form a complex that assembles clathrin more efficiently than either protein alone. Phosphorylation of AP180, by modulating the affinity of AP180 for AP-2, may contribute to the regulation of clathrin assembly in vivo.

Clathrin - Protein interactions

There is a complex network of protein–protein and protein–lipid interactions that underlie clathrin‐mediated vesicular traffic in all compartmentalized cells from yeast to man. Major progress has been made in the determination of the three‐dimensional structures of many of the components. Recently, there has been an explosion in the identification and characterization of clathrin binding partners. This review integrates the structural and biochemical information that is currently available to present a unified view of how many clathrin binding partners interact with clathrin.

Uncoating of Clathrin-Coated Vesicles in Presynaptic Terminals: Roles for Hsc70 and Auxilin

We have examined the roles of Hsc70 and auxilin in the uncoating of clathrin-coated vesicles (CCVs) during neuronal endocytosis. We identified two peptides that inhibit the ability of Hsc70 and auxilin to uncoat CCVs in vitro. When injected into nerve terminals, these peptides inhibited both synaptic transmission and CCV uncoating. Mutation of a conserved HPD motif within the J domain of auxilin prevented binding to Hsc70 in vitro and injecting this mutant protein inhibited CCV uncoating in vivo, demonstrating that the interaction of auxilin with Hsc70 is critical for CCV uncoating. These studies establish that auxilin and Hsc70 participate in synaptic vesicle recycling in neurons and that an interaction between these proteins is required for CCV uncoating.

Regulation of AP-3 Function by Inositides IDENTIFICATION OF PHOSPHATIDYLINOSITOL 3,4,5-TRISPHOSPHATE AS A POTENT LIGAND

As part of the growing effort to understand the role inositol phosphates and inositol lipids play in the regulation of vesicle traffic within nerve terminals, we determined whether or not the synapse-specific clathrin assembly protein AP-3 can interact with inositol lipids. We found that soluble dioctanoyl-phosphatidylinositol 3,4,5-trisphosphate (DiC8PtdIns(3,4,5)P3) was only 7.5-fold weaker a ligand than D-myo-inositol hexakisphosphate in assays that measured the displacement of D-myo-[3H]inositol hexakisphosphate. In functional assays we found that both of these ligands inhibited clathrin assembly, but DiC8-PtdIns(3,4,5)P3 was more potent and exhibited a larger maximal effect. We also examined the structural features of DiC8-PtdIns(3,4,5)P3 that establish specificity. Dioctanoyl-phosphatidylinositol 3,4-bisphosphate, which does not have a 5-phosphate, and 4,5-O-bisphosphoryl-D-myo-inosityl 1-O-(1,2-O-diundecyl)-sn-3-glycerylphosphate, which does not have a 3-phosphate, were, respectively, 2-fold and 4-fold less potent than DiC8-PtdIns(3,4,5)P3 as inhibitors of clathrin assembly. Deacylation of DiC8-PtdIns(3,4,5)P3 reduced its affinity for AP-3 almost 20-fold, and also dramatically lowered its ability to inhibit clathrin assembly. The deacylated products of the soluble derivatives of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 4,5-bisphosphate were both not significant inhibitors of clathrin assembly. It therefore appears that the interactions of inositides with AP-3 should not be considered simply in terms of electrostatic effects of the highly charged phosphate groups. Ligand specificity appears also to be mediated by hydrophobic interactions with the fatty-acyl chains of the inositol lipids.

A Role for the Clathrin Assembly Domain of AP180 in Synaptic Vesicle Endocytosis

We have used the squid giant synapse to determine whether clathrin assembly by AP180 is important for synaptic vesicle endocytosis. The squid homolog of AP180 encodes a 751 amino acid protein with 40% sequence identity to mouse AP180. Alignment of squid AP180 with other AP180 homologs shows that amino acid identity was highest in the N-terminal inositide-binding domain of the protein and weakest in the C-terminal clathrin assembly domain. Recombinant squid AP180 was able to assemble clathrin in vitro, suggesting a conserved three-dimensional structure that mediates clathrin assembly despite the divergent primary sequence of the C-terminal domain. Microinjection of the C-terminal domains of either mouse or squid AP180 into the giant presynaptic terminal of squid enhanced synaptic transmission. Conversely, a peptide from the C-terminal domain of squid AP180 that inhibited clathrin assembly in vitro completely blocked synaptic transmission when it was injected into the giant presynaptic terminal. This inhibitory effect occurred over a time scale of minutes when the synapse was stimulated at low (0.03 Hz), physiological rates. Electron microscopic analysis revealed several structural changes consistent with the inhibition of synaptic vesicle endocytosis; peptide-injected terminals had far fewer synaptic vesicles, were depleted of coated vesicles, and had a larger plasma membrane perimeter than terminals injected with control solutions. In addition, the remaining synaptic vesicles were significantly larger in diameter. We conclude that the clathrin assembly domain of AP180 is important for synaptic vesicle recycling at physiological rates of activity and that assembly of clathrin by AP180 is necessary for maintaining a pool of releasable synaptic vesicles.