Selective Targeting of a Novel Epsin–VEGFR2 Interaction Promotes VEGF-Mediated Angiogenesis

Abstract

Rationale We previously reported that VEGF-induced binding of VEGFR2 to epsins 1 and 2 triggers VEGFR2 degradation and attenuates VEGF signaling. The epsin ubiquitin interacting motif (UIM) was shown to be required for the interaction with VEGFR2, however the molecular determinants that govern how epsin specifically interacts with and regulates VEGFR2 were unknown. Objective The goals for the present study were (1) to identify critical molecular determinants that drive the specificity of the epsin and VEGFR2 interaction and (2) to ascertain if such determinants were critical for physiological angiogenesis in vivo. Methods and Results Structural modeling uncovered two novel binding surfaces within VEGFR2 that mediate specific interactions with epsin UIM. Three glutamic acid residues in epsin UIM were found to interact with residues in VEGFR2. Further, we found that the VEGF-induced VEGFR2-epsin interaction promoted c-Cbl-mediated ubiquitination of epsin, and uncovered a previously unappreciated ubiquitin-binding surface within VEGFR2. Mutational analysis revealed that the VEGFR2-epsin interaction is supported by VEGFR2 interacting specifically with the UIM and with ubiquitinated epsin. An epsin UIM peptide, but not a mutant UIM peptide, potentiated endothelial cell proliferation, migration and angiogenic properties in vitro, increased postnatal retinal angiogenesis, and enhanced VEGF-induced physiological angiogenesis and wound healing. Conclusions Distinct residues in the epsin UIM and VEGFR2 mediate specific interactions between epsin and VEGFR2, in addition to UIM recognition of ubiquitin moieties on VEGFR2. These novel interactions are critical for pathophysiological angiogenesis, suggesting that these sites could be selectively targeted by therapeutics to modulate angiogenesis.

Endothelial epsins as regulators and potential therapeutic targets of tumor angiogenesis

“…Interestingly, c-Cbl-dependent ubiquitination of epsin increases in response to VEGF stimulation to further promote the interaction between epsin and VEGFR2. The two residues identified as novel interfaces for binding to epsin 1, H891, and S1021 show potential in interacting with ubiquitin [48]. In this scenario, residues E183, E184, and E185 determine the classical function of UIM to bind with VEGFR2, while the novel ubiquitin-interacting-interface of the VEGFR2 kinase domain binds with ubiquitinated epsins to thereby reinforce interactions between the two.…”

“…In this scenario, residues E183, E184, and E185 determine the classical function of UIM to bind with VEGFR2, while the novel ubiquitin-interacting-interface of the VEGFR2 kinase domain binds with ubiquitinated epsins to thereby reinforce interactions between the two. Remarkably, the study also demonstrates in vitro and in vivo activity of peptide-based angiogenesis modulators targeting this interaction [48]. …”

Therapeutic efficacy of a synthetic epsin mimetic peptide in glioma tumor model: uncovering multiple mechanisms beyond the VEGF-associated tumor angiogenesis

“…Ubiquitin interacting motifs (UIMs) located at the downstream of ENTH domain in the N-terminal of the protein help to promote the interaction between epsin and VEGFR2, thus promoting endocytosis [25]. UIM is a 18-sequence motif that is able to specifically detect and bind to ubiquitylated VEGFR2 through specific residues on UIM and VEGFR2 respectively [26, 27]. After binding to the ubiquitylated VEGFR2, it acts as a type of cargo carrier, which promotes epsin binding [26, 27].…”

“…UIM is a 18-sequence motif that is able to specifically detect and bind to ubiquitylated VEGFR2 through specific residues on UIM and VEGFR2 respectively [26, 27]. After binding to the ubiquitylated VEGFR2, it acts as a type of cargo carrier, which promotes epsin binding [26, 27]. Epsin binds to the activated VEGFR2 and degrades it, which eventually causes VEGFR2 to break down into endosomes and lysosomes, leaving only parts of VEGFR2 remaining.…”

Ubiquitination and Phosphorylation are Independently Required for Epsin-Mediated Internalization of Cargo inS. cerevisiae

“…Indeed, the complexity of the trafficking information packed in the cytosolic (and possibly transmembrane) portions of membrane proteins should not be underestimated. Important work done in the Chen lab clearly indicated that in mammalian epsins specificity determinants for the recognition of the receptor tyrosine kinase VEGFR2 are embedded within the UIM sequence environment(78)(79)(80). Further, it should be kept in mind that multi-spanning proteins like Ena1 usually contain additional/critical determinants in their intracellular loops; these regions may play a role in the recruitment of post-translational modifying enzymes; e.g., kinases, ubiquitin ligases/ARTs; or proteins that may assist in cargo recognition.…”