No abstract

Although interactions between the μ2 subunit of the clathrin adaptor protein complex AP-2 and tyrosinebased internalization motifs have been implicated in the selective recruitment of cargo molecules into coated pits, the functional significance of this interaction for endocytosis of many types of membrane proteins remains unclear. To analyze the function of μ2-receptor interactions, we constructed an epitope-tagged μ2 that incorporates into AP-2 and is targeted to coated pits. Mutational analysis revealed that Asp176 and Trp421 of μ2 are involved in the interaction with internalization motifs of TGN38 and epidermal growth factor (EGF) receptor. Inducible overexpression of mutant μ2, in which these two residues were changed to alanines, resulted in metabolic replacement of endogenous μ2 in AP-2 complexes and complete abrogation of AP-2 interaction with the tyrosine-based internalization motifs. As a consequence, endocytosis of the transferrin receptor was severely impaired. In contrast, internalization of the EGF receptor was not affected. These results demonstrate the potential usefulness of the dominant-interfering approach for functional analysis of the adaptor protein family, and indicate that clathrin-mediated endocytosis may proceed in both a μ2-dependent and -independent manner.

We demonstrate that PS-341, a small molecule inhibitor of the proteasome, markedly sensitizes resistant prostate, colon, and bladder cancer cells to TNF-like apoptosisinducing ligand (TRAIL)-induced apoptosis irrespective of Bcl-xL overexpression. PS-341 treatment by itself does not affect the levels of Bax, Bak, caspases 3 and 8, c-Flip or FADD, but elevates levels of TRAIL receptors DR4 and DR5. This increase in receptor protein levels is associated with the ubiquitination of the DR5 protein.When PS-341 is combined with TRAIL, the levels of activated caspase 8 and cleaved Bid are substantially increased. In Bax-negative TRAIL-resistant HC-4 colon cancer cells, the combination of PS-341 and TRAIL overcomes the block to activation of the mitochondrial pathway and causes SMAC and cytochrome c release followed by apoptosis. Similarly, murine embryonic fibroblasts lacking Bax undergo apoptosis when exposed to the combination of PS-341 and TRAIL; however, fibroblasts lacking Bak are significantly resistant. Taken together, these findings indicate that PS-341 enhances TRAIL-induced apoptosis by increasing the cleavage of caspase 8, causing Bak-dependent release of mitochondrial proapoptotic proteins.

Defining the molecular targets of insecticides is crucial for assessing their selectivity and potential impact on environment and health. Two commercial insecticides are now shown to target a transient receptor potential (TRP) ion channel complex that is unique to insect stretch receptor cells. Pymetrozine and pyrifluquinazon disturbed Drosophila coordination and hearing by acting on chordotonal stretch receptor neurons. This action required the two TRPs Nanchung (Nan) and Inactive (Iav), which co-occur exclusively within these cells. Nan and Iav together sufficed to confer cellular insecticide responses in vivo and in vitro, and the two insecticides were identified as specific agonists of Nan-Iav complexes that, by promoting cellular calcium influx, silence the stretch receptor cells. This establishes TRPs as insecticide targets and defines specific agonists of insect TRPs. It also shows that TRPs can render insecticides cell-type selective and puts forward TRP targets to reduce side effects on non-target species.

Arrays promise to advance biology through parallel screening for binding partners. We show the combinatorial in situ synthesis of 40,000 peptide spots per square centimeter on a microchip. Our variant Merrifield synthesis immobilizes activated amino acids as monomers within particles, which are successively attracted by electric fields generated on each pixel electrode of the chip. With all different amino acids addressed, particles are melted at once to initiate coupling. Repetitive coupling cycles should allow for the translation of whole proteomes into arrays of overlapping peptides that could be used for proteome research and antibody profiling.