Recently identified trace amine receptors are potential direct targets for drugs of abuse, including amphetamine and 3,4-methylenedioxymethamphetamine (MDMA). We cloned fulllength rhesus monkey trace amine receptor 1 (rhTA 1 ) that was 96% homologous to human TA 1 . The trace amines tyramine and ␤-phenylethylamine (PEA) and the monoamine transporter substrates (Ϯ)-amphetamine and (Ϯ)-MDMA stimulated cAMP accumulation in rhTA 1 -expressing cell lines, as measured by a cAMP response element-luciferase assay. Cocaine did not stimulate cAMP accumulation in rhTA 1 cells, but it blocked [ 3 H]PEA transport mediated by the dopamine transporter. Cotransfection with the human dopamine transporter enhanced PEA-, amphetamine-, and MDMA-mediated rhTA 1 receptor activation, but it diminished tyramine activation of rhTA 1 . Because TA 1 (EGFP-rhTA 1 chimera) was largely intracellular, conceivably the dopamine transporter can facilitate access of specific agonists to intracellular TA 1 . rhTA 1 mRNA expression was detected in rhesus monkey substantia nigra, implying that TA 1 may be colocalized with the dopamine transporter in dopamine neurons. In summary, primate TA 1 receptors are direct targets of trace amines, amphetamine, and MDMA. These receptors could also be indirect targets of amphetamine, MDMA, and cocaine through modification of monoamine transporter function. Conceivably, rhTA 1 receptors may be located on pre-or postsynaptic membranes. Interference with the carrier function of monoamine transporters with a consequent rise of extracellular levels of trace amines could activate these receptors. The cloning of a highly homologous TA 1 from rhesus monkey and demonstration that rhTA 1 receptors are activated by drugs of abuse, indicate that nonhuman primates may serve to model physiological and pharmacological TA 1 -mediated responses in humans.

Roundabouts (robo) are cell-surface receptors that mediate repulsive signaling mechanisms at the central nervous system midline. However, robos may also mediate attraction mechanisms in the context of vascular development. Here, we have performed structure-function analysis of roundabout4 (Robo4), the predominant robo expressed in embryonic zebrafish vasculature and found by gain of function approaches in vitro that Robo4 activates Cdc42 and Rac1 Rho GTPases in endothelial cells. Indeed, complementary robo4 gene knockdown approaches in zebrafish embryos show lower amounts of active Cdc42 and Rac1 and angioblasts isolated from these knockdown embryos search actively for directionality and guidance cues. Furthermore, Robo4-expressing endothelial cells show morphology and phenotype, characteristic of Rho GTPase activation. Taken together, this study suggests that Robo4 mediates attraction-signaling mechanisms through Rho GTPases in vertebrate vascular guidance. Roundabouts (Robos)4 are class of neural guidance receptors that bind to the slit family of guidance cues and primarily mediate axon repulsion signals (1, 2). Outside the nervous system, slit-robo signaling has been implicated in inhibition of leukocyte migration (3), kidney induction (4), and vascular system (5, 6). In vertebrates, three Robo receptor family members were identified, all with prominent neural expression (7,8), whereas robo4 is the predominant robo expressed in the embryonic vasculature during development (6). Previously, we have shown that robo4 is essential for angiogenesis in vivo and that robo4 knockdown embryos display spatial and temporal defects in intersomitic vessels (ISVs) sprouting (9). Two recent reports have begun addressing the nature of downstream signaling molecules triggered by Robo4. One reports mammalian enabled protein interaction with Robo4 cytoplasmic tail (6), and another reports that overexpression of Robo4 in endothelial cells down-regulates ERK and FAK (10). To date, no report has identified the structural requirements for these activities of Robo4. To study signaling mechanisms triggered by Robo4 in the vasculature, we initially designed four structural mutants tagged with GFP. Overexpression analysis in zebrafish in vivo and in HEK-293T or porcine aortic endothelial (PAE) cells in vitro was used to determine the functionality of the constructs, and signaling pathways triggered by the mutant proteins. Pull-down analysis in transfected HEK-293T and endothelial cells show that full-length Robo4 activate Cdc42 and Rac1 but not Rho and that this activation was dependent on the presence of the extracellular ligand-binding region in Robo4. Furthermore, Rho GTPase activation is conserved across other family members in evolution and correlates well with phenotypic morphological changes in Robo4 expressing endothelial cell such as the induction of filopodia and lamellipodia. In addition, robo4 knockdown embryos in vivo display lower amounts of active Cdc42 and Rac, and angioblasts isolated ex vivo from robo4 knockdown transg...

Phosphatidylinositol-3-kinase (PI3K), and its downstream effector Akt, or protein kinase B␣ (PKB␣), play a major regulatory role in control of apoptosis, proliferation, and angiogenesis. PI3K and Akt are amplified or overexpressed in a number of malignancies, including sarcomas, ovarian cancer, multiple myeloma, and melanoma. This pathway regulates production of the potent angiogenic factor vascular endothelial growth factor (VEGF), and protects tumor cells against both chemotherapy and reactive oxygen-induced apoptosis through phosphorylation of substrates such as apoptotic peptidaseactivating factor-1 (APAF-1), forkhead proteins, and caspase 9. Given its diverse actions, compounds that suppress the PI3K/Akt pathway have potential pharmacologic utility as angiogenesis inhibitors and antineoplastic agents. Using the SVR angiogenesis assay, a screen of natural products, we isolated the alkaloid solenopsin, and found that it is a potent angiogenesis inhibitor. We also found that solenopsin inhibits the PI3K signaling pathway in IntroductionThe serine/threonine kinase c-Akt-1, or protein kinase B␣ (PKB), is the cellular homolog of a transforming oncogene initially isolated from a lymphoma. Akt is a downstream target of phosphatidylinositol-3-kinase (PI3K), a family of at least 4 different enzymes, with the prototypical PI3K heterodimer consisting of a p85 (regulatory) and a p110 (catalytic) subunit. The PI3K/Akt pathway is involved in the regulation of diverse cellular functions including proliferation, cytoskeletal organization, survival, and malignant transformation. [1][2][3][4] Upon binding of PI3K products to its pleckstrin homology domain, Akt is translocated to the plasma membrane where it is activated by upstream phosphorylated kinases, including PI3K-dependent kinases 1 and 2 (PDK1 and PDK2) and mammalian target of rapamycin complex 2 (mTORC2). The PI3K/Akt pathway is stimulated by numerous receptor tyrosine kinases and oncogenes, including receptors for insulin-like growth factor 1 (IGF-1), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), ras, Her2/neu, and polyoma middle T oncogenes. [5][6][7][8][9][10] Because Akt plays a central role in regulating apoptosis, angiogenesis, and metabolism of cells, Akt is an attractive pharmacologic target for the treatment of cancer and inflammation. 11,12 Small-molecular-weight inhibitors of PI3K include LY 294002 and the fungal metabolite wortmannin, 13 as well as ether phospholipids, including perifosine, which has entered clinical trials. 14,15 Using the SVR angiogenesis assay, [16][17][18] we found that solenopsin A, 19,20 the primary alkaloid from the fire ant Solenopsis invicta, has antiangiogenic activity. We also discovered that solenopsin disrupted angiogenesis in vivo in embryonic zebrafish. In order to determine the mode of action, we examined the ability of solenopsin to inhibit a battery of cellular kinases and found that solenopsin A inhibited Akt relatively selectively in an ATP-competitive manner without affecting its u...