Signal peptides and growth factor signaling

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Signal sequences target newly translated proteins to specific locations within and outside the cell, and accordingly, play a key role in cell biology. Beyond their classical targeting functions, a number of non- canonical functions of specific signal peptides have been reported in recent years, mostly in lower organisms, but also in eucaryotic cells. However, much remains to be learned regarding the biology of signal sequences, which have emerged as a new focus for drug development. Vascular endothelial growth factor (VEGF) is the prototypic growth factor in angiogenesis, a key process during development, growth, adult homeostasis, and response to injury, which is dysregulated in a broad spectrum of diseases, including ischemia-related cardiovascular diseases, diabetic retinopathy and malignancy. The VEGF effects are primarily mediated by its high affinity binding to VEGF receptor (VEGFR)-2 which triggers activation of several downstream signaling pathways. This is a tightly controlled process with multiple layers of regulation to ensure a finely tuned signal. Endothelial and smooth muscle cell-derived neuropilin-like protein (DCBLD2) is an integral membrane protein with an unusually long, two-subdomain organization signal sequence. We have identified DCBLD2 as a regulator of VEGF signaling and developmental and ischemic angiogenesis. DCBLD2 associates with VEGFR- 2 and regulates VEGF signaling by modulating VEGFR-2-VE-cadherin-protein tyrosine phosphatase complex formation. In search of DCBLD2 domain(s) involved in its interaction with VEGFR-2, unexpectedly we identified a functional interaction between the DCBLD2 signal sequence and VEGFR-2. DCBLD2 signal sequence expression, or addition of a synthetic peptide encompassing its hydrophobic, C-terminal moiety to cultured cells promoted VEGF signal transduction. This led us to hypothesize that DCBLD2 signal sequence regulates VEGF (and possibly other growth factors’) signal transduction and modulates angiogenesis, also raising the possibility that other similarly structured signal sequences may play a regulatory role in growth factor signaling. Here, we seek to expand the scope of these observations to define, and determine the mechanism of action of, the post-targeting functions of DCBLD2 signal sequence on VEGF and related signaling pathways. To this end, our specific aims are to investigate non-canonical functions of DCBLD2 signal sequence in VEGF signal transduction and evaluate the effects of exogenous DCBLD2 signal sequence-related peptides on VEGF signaling and angiogenesis. The proposed experiments establish a novel post-targeting function for signal sequences in growth factor signal transduction and set the stage for future development of signal sequence- based therapeutics, not only for angiogenesis, but potentially other disorders.