A signal sequence multi-leucine motif regulates VEGF receptor signaling

Signal sequences (SS) target newly synthesized proteins to the endoplasmic reticulum and, eventually, the plasma membrane. Discoidin, CUB, and LCCL domain containing 2 (DCBLD2) is a transmembrane protein with an unusually long SS that interacts with vascular endothelial growth factor receptor (VEGFR)-2 and promotes VEGF-induced endothelial cell signaling, proliferation and migration, as well as angiogenesis. How DCBLD2 interacts with VEGFR-2 to modulate VEGF signaling remains unclear. We generated various constructs containing different combinations of DCBLD2 domains, including its SS, and conducted co-immunoprecipitation and signaling studies in HEK 293T and endothelial cells to identify the smallest DCBLD2 unit that interacts with VEGFR-2. Other proteins containing this unit were identified, and their interactions with VEGFR-2 were evaluated. The DCBLD2 SS interacted with VEGFR-2 and promoted VEGF signaling. The smallest unit in the DCBLD2 SS that interacted with VEGFR-2 was the L5VL5 sequence. Even after the central valine was removed, the L10 sequence mimicked the DCBLD2 SS effect on VEGF-signaling, while shorter or longer multi-leucine sequences were less effective. We identified several other human proteins containing such uncleaved multi-leucine SS, including CD45-associated protein (CD45-AP) and BRICHOS domain containing 5 (BRICD5). Both these proteins could interact with VEGFR-2, and CD45-AP could modulate VEGF signaling in endothelial cells. In conclusion, a multi-leucine motif in SS can directly engage in protein-protein interactions and modulate VEGF signaling. These findings refine the traditional view of SS as merely targeting elements, emphasizing their role in cellular signaling and opening new avenues for research and therapy.