Defective angiogenesis underlies the pathogenesis of over 50 malignant, ischemic and inflammatory diseases. While current antagonists of VEGF signaling have a wide range of therapeutic applications, most of these treatments fail to provide long-term efficacy due to acquired resistance and toxicities. Overcoming these clinical challenges will therefore require addressing a number of critical aspects of VEGF signaling that are very much unclear. VEGFR2 is the principal driver of sprouting angiogenesis as its membrane trafficking controls the specificity, duration and amplitude of many, if not most, of the VEGF-induced signaling pathways. But unlike the molecular basis of VEGFR2 tyrosine phosphorylation that transduces receptor signaling, how its membrane trafficking and turnover are spatiotemporally coordinated by various serine/threonine kinases, chaperones and ubiquitin ligases remain poorly understood. In fact, although PKCs have been long recognized as key mediators of VEGFR2 degradation, it is still unclear whether PKCs directly or indirectly promote serine/threonine phosphorylation-induced turnover. Here our work supports an exciting new mechanism by which VEGFR2 stability is regulated through a novel membrane-based signaling complex. In preliminary studies, we discovered that ?IV-spectrin, a large membrane cytoskeletal scaffolding protein characterized only in the nervous system and heart, is expressed in vascular endothelial cells (ECs) to act as a critical negative regulator of angiogenesis. ?IV-spectrin dysfunction in newborn mice and zebrafish embryos produce debilitating hypersprouting vessels in part due to abnormally high levels of VEGF/VEGFR2 signaling and dramatically elevated number of tip cells. Our data strongly suggest that ?IV-spectrin functions as a crucial signaling platform by which VEGFR2 is targeted for degradation through direct CaMKII-induced phosphorylation of novel serine/threonine sites. Based on our findings, we hypothesize that the ?IV-spectrin/CaMKII signaling complex regulates VEGFR2 phosphorylation, membrane trafficking and turnover to suppress VEGF signaling and tip cell phenotype during sprouting angiogenesis. Two aims are proposed to test this hypothesis: 1) Define ?IV- spectrin-based mechanisms of VEGF signaling during sprouting angiogenesis; 2) Establish the role of ?IV- spectrin in endothelial tip and stalk cell specification. Collectively, results from these studies will address a crucial question in VEGFR signaling through the characterization of a novel ?IV-spectrin signaling complex, and identify new vascular targets in failed long-term VEGF-related therapies.