Physical and functional interaction between the dopamine transporter and the synaptic vesicle protein synaptogyrin-3.

Uptake through the dopamine transporter (DAT) represents the primary mechanism used to terminate dopaminergic transmission in brain. Although it is well known that dopamine (DA) taken up by the transporter is used to replenish synaptic vesicle stores for subsequent release, the molecular details of this mechanism are not completely understood. Here, we identified the synaptic vesicle protein synaptogyrin-3 as a DAT interacting protein using the split ubiquitin system. This interaction was confirmed through coimmunoprecipitation experiments using heterologous cell lines and mouse brain. DAT and synaptogyrin-3 colocalized at presynaptic terminals from mouse striatum. Using fluorescence resonance energy transfer microscopy, we show that both proteins interact in live neurons. Pull-down assays with GST (glutathione S-transferase) proteins revealed that the cytoplasmic N termini of both DAT and synaptogyrin-3 are sufficient for this interaction. Furthermore, the N terminus of DAT is capable of binding purified synaptic vesicles from brain tissue. Functional assays revealed that synaptogyrin-3 expression correlated with DAT activity in PC12 and MN9D cells, but not in the non-neuronal HEK-293 cells. These changes were not attributed to changes in transporter cell surface levels or to direct effect of the protein-protein interaction. Instead, the synaptogyrin-3 effect on DAT activity was abolished in the presence of the vesicular monoamine transporter-2 (VMAT2) inhibitor reserpine, suggesting a dependence on the vesicular DA storage system. Finally, we provide evidence for a biochemical complex involving DAT, synaptogyrin-3, and VMAT2. Collectively, our data identify a novel interaction between DAT and synaptogyrin-3 and suggest a physical and functional link between DAT and the vesicular DA system.

Structural studies of proteinase entrapment by human alpha 2-macroglobulin using 3-D electron microscopy

Human a2 -macroglobulin ( a2 M; homotetramer, Mr 720 kDa) is an essential scavenger of proteinases in the serum. Each of its four subunits has a ‘bait region’, with cleavage sequences for almost all endo-proteinases, an unusual thiol ester moiety and a receptor-binding domain (RBD). Bait region cleavage in native a2 M ( a2 M-N) by a proteinase results in rapid thiol ester breakage, with a large-scale structural transformation, in which a2 M uniquely entraps the proteinase in a cage-like structure and exposes receptor-binding domains for rapid endocytosis. Transformed a2 M ( a2 M-TR) contains up to two proteinases, which remain active to small substrates. 3-D electron microscopy is optimally suited to study this unusual structural change at resolutions near (1/30) Å−1. ^ The structural importance of the thiol esters was demonstrated by a genetically-engineered a2 M, with the cysteines involved in thiol ester formation mutated to serines, which appeared structurally homologous to a2 M-TR. This demonstrates that the four highly labile thiol esters alone maintain the a2 M-N structure, while the ‘closed trap’ formed by a2 M-TR is a more stable structural form. ^ Half-transformed a2 M ( a2 M-HT), with cleaved bait regions and thiol esters in only two of its four subunits, provides an important structural link between a2 M-N and a2 M-TR. A comparison with a2 M-N showed the two proteinase-entrapping domains were above and below the plane bisecting the long axis. Both a2 M-N and a2 M-TR consist of two dense, oppositely twisted strands with significant interconnections, indicating that the structural change involves a rotation of these strands. In a2 M-HT these strands were partially untwisted with large central openings, revealing the manner in which the proteinase enters the internal cavity of a2 M. ^ In reconstructions of a2 M-N, a2 M-HT and a2 M-TR labeled with a monoclonal Fab, the Fabs were located on distal ends of each constitutive strand, demonstrating an anti-parallel arrangement of the subunits. Separation between the top and bottom pairs of Fabs was nearly the same on all structures, but the pairs were rotated about the long axis. Taken together, these results indicate that upon proteinase cleavage the two strands in a2 M-N separate. The proteinase enters the structure, while the strands re-twist to encage it. In a2 M-TR, which displays receptor-binding arms, more than two subunits are transformed as strands in the transformed half of a2 M-HT were not separated. ^

A novel mechanism of regulation of phosphatidylinositol 3 -kinase: Tyrosine phosphorylation of p85 residue 688

Phosphatidylinositol 3-kinase (PI3K) phosphorylates membrane constituent phosphatidylinositols, producing second messengers that link membrane bound receptor signals to cellular proliferation and survival. PI3K, a heterodimer consisting of a catalytic p110 subunit and a regulatory p85 subunit, can be activated through induced association with other signaling molecules. The p85 subunit serves to both stabilize and inactivate p110. The inhibitory activity of P85 is relieved by occupancy of the N terminal SH2 domain by phosphorylated tyrosine. PI3K becomes phosphorylated and activated subsequent to a variety of stimuli. Indeed, Src family kinases have been demonstrated to phosphorylate p85 at tyrosine 688, but the role of phosphorylation in PI3K function is unclear. We decided to evaluate the importance of tyrosine phosphorylation to PI3K activity. We demonstrate that tyrosine phosphorylated p85 is associated with a higher specific activity than is non-phosphorylated PI3K. Wild type p85 inhibits PI3K enzyme activity, a process accentuated by mutation of tyrosine 688 to alanine and reversed by mutation to aspartate which functions as a phosphotyrosine mimic in multiple systems. Strikingly, the Y688D mutation completely reverses the p85 inhibitory activity on cell viability and activation of downstream protein NFkB. We demonstrate that tyrosine phosphorylated Y688 or Y688D is sufficient to bind the p85 N terminal SH2 domain, either within full length p85 or in an isolated N terminal SH2 domain, suggesting the possibility of an intramolecular interaction between phosphorylated Y688 and the p85 N terminal SH2 domain that can relieve the p85-induced inhibition of p110. Further, we provide evidence that dephosphorylation of Y688 reduces phosphorylation-induced PI3K activity. We demonstrate that tyrosine phosphatase SHP-1 can physically associate with p85 in a SH2-mediated interaction with the C terminal tail of SHP-1. This association is concomitant with both p85 dephosphorylation and decreased PI3K activity. Altogether, our data suggests the phosphorylation state of p85 is the focal point of a novel mechanism for PI3K activity regulation. As PI3K has been shown to be involved in the vital physiological processes of cell proliferation and apoptosis, a thorough understanding of the regulation of this signaling protein may provide opportunities for the design of novel treatments for cancer. ^