Domain characterization and intracellular trafficking of a novel protein, Carom, and its role in VEGF receptor endocytosis

Abstract

The novel protein Carom is a homolog of the Drosophila protein, Nervous Wreck (Nwk). Carom was cloned as a homocysteine response protein in primary endothelial cells (EC). In order to discern the functionality of the protein in higher order mammals and potential role in homocysteine-inhibited angiogenesis, we performed basic characterization studies on Carom. Theoretical modeling of Carom matched the solved structure of Nwk. Using a lab-generated antibody against Carom’s F-BAR domain, it is evident that Carom is localized to the mitochondria and speckling in the nucleus of primary ECs. In order to perform biochemical and structural studies in primary ECs, Carom was cloned from an adenoviral shuttle vector to an adeno-associated virus (AAV) transfer vector. We created a multi-cistronic open reading frame with an N-terminal Flag and a cleavable C-terminal green fluorescent protein (mClover3). Primary ECs are difficult to transfect, so we optimized an AAV packaging system in order to get high titers and high purity AAV that can transduce our primary ECs. Carom is composed of several functional domains with the capability of binding to cell membranes and act as a scaffolding for attaching adaptor proteins. To isolate which domains are important to the partner binding and cellular localization, we serially truncated the domains from Carom starting from both the C-terminus and N-terminus. We demonstrated that the C-terminal region features some post-translational modifications creating the second band in western blots with lower mobility. Also, the F-BAR domain is responsible for translocalization of Carom from the cytoplasm to the cell membrane and nucleus. A novel mechanism is proposed for why Carom is upregulated in response to homocysteine (Hcy), an independent risk factor for cardiovascular disease. It is previously known that Hcy inhibits angiogenesis. Our data mining studies identified a potentially important receptor for angiogenesis in ECs, VEGFR2, being endocytosed and ultimately degraded. Through biotinylation assays, we determined that Carom does help enhance the endocytosis of VEGFR2 potentially leading to degradation via the lysosome. In summary, Carom is endogenously localized to the mitochondria in primary ECs, the C-terminus is post-translationally modified, the bipartite nuclear localization signal containing F-BAR domain localizes to the cell membrane and nucleus, and Carom enhances the endocytosis of VEGFR2.