Abstract
Wg/Wnt as a signaling protein binds to Frizzled (Fz) and Arrow (Arr), two Wg co-receptors essential for Wg signaling for cell proliferation, differentiation, and cell survival. Arr has a long extracellular region, a single transmembrane domain and an intracellular region. Here, we report that a new arrm7 mutant is identified in a genetic screen as a suppressor of lethality induced by overexpression of Sol narae (Sona), a secreted metalloprotease in ADAMTS family involved in Wg signaling. arrm7 allele has a premature stop codon, which encodes Arrm7 protein missing the intracellular region. arrm7 clones show cell death phenotype and overexpression of Arrm7 protein also induces cell death. Levels of extracellular Sona were decreased in both arrm7 and arr2 null clones, demonstrating that Arr increases the level of extracellular Sona. Indeed, Arr but not Arrm7, increased levels of Sona in cytoplasm and exosome fraction by inhibiting the lysosomal degradation pathway. Interestingly, Arr itself was identified in the exosome fraction, demonstrating that Arr is secreted to extracellular space. When Sona-expressing S2 cells were treated with exosomal Arr, the extracellular level of active Sona was increased. These results show that exosomal Arr dictates Sona-expressing cells to increase the level of extracellular Sona. This new function of Arr occurred in the absence of Wg because S2 cells do not express Wg. We propose that Arr plays two distinct roles, one as an exosomal protein to increase the level of extracellular Sona in a Wnt-independent manner and the other as a Wg co-receptor in a Wnt-dependent manner.
Highlights
Wnt signaling is an important pathway conserved in all metazoans for cell proliferation, cell survival, and differentiation[1,2,3,4]
We propose that Arr plays two distinct roles, one as an exosomal protein to increase the level of extracellular Sol narae (Sona) in a Wnt-independent manner and the other as a Wg co-receptor in a Wnt-dependent manner
We report here that the loss of arr decreases the level of extracellular Sona in flies, and Arr increases the extracellular level of Sona by stabilizing intracellular Sona in Schneider 2 (S2) cells through inhibition of lysosomal pathway
Summary
Fly lines arrm[7] mutant was derived from m7 sona suppressor as described[16]. UAS-arrm7-HA, FRT42D arrm[7], and FRT42D arr[2] are generated with arrm[7] and BDSC #3287 for this study. The obtained P100 fraction contains EVs including exosomes, was washed with PBS, and pelleted again by additional centrifugation at 100,000×g. Antibodies were diluted in wash buffer and incubated overnight at 4 °C or 2 h at room temperature. Larvae were dissected in icecold M3 medium and incubated in M3 medium containing primary antibody for 2 h at 4 °C as described[31]. Samples were processed by the same way as described above but buffers did not contain any detergent. For co-IP, protein G agarose beads (Merck Millipore) was incubated in primary antibody containing lysis buffer (20 mM HEPES, 150 mM NaCl, 2 mM DTT, 5 mM EDTA, 5 mM EGTA, 0.2% Triton X-100, 10% glycerol and Roche complete protease inhibitor cocktail) for 2 h at room temperature. Beads were washed with the lysis buffer several times and used for western analysis
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