CCM2-CCM3 interaction stabilizes their protein expression and permits endothelial network formation.
Bottom Line: Mutations in the essential adaptor proteins CCM2 or CCM3 lead to cerebral cavernous malformations (CCM), vascular lesions that most frequently occur in the brain and are strongly associated with hemorrhagic stroke, seizures, and other neurological disorders.CCM2 binds CCM3, but the molecular basis of this interaction, and its functional significance, have not been elucidated.However, CCM3 expression in the absence of CCM2 is sufficient to support normal cell growth, revealing complex-independent roles for CCM3.
Affiliation: Department of Pharmacology and Department of Cell Biology, Yale University, New Haven, CT 06520.Show MeSH
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Mentions: To understand the functional significance of the CCM2–CCM3 interaction, we first sought a detailed characterization of the CCM2–CCM3 interface. CCM2 contains an N-terminal PTB domain (Liquori et al., 2003; Fisher et al., 2015), followed by a linker region and an HHD (Fisher et al., 2013; Fig. 1 A), but the portion of CCM2 that binds to CCM3 has not previously been mapped. To probe the minimal region of CCM2 that could bind CCM3, we therefore conducted pull-down assays using various N-terminally tagged GST fusion constructs of CCM2 bound to glutathione Sepharose beads and purified, soluble N-terminally 6×His-tagged full-length CCM3. Bound CCM3 was detected by immunoblotting. We tested a nearly full-length protein (residues 1–438; CCM2FL), a PTB domain construct (residues 51–251) that includes a significant portion of the linker region between the PTB and HHD domains (CCM2PTB-LongLinker, CCM2PTB-LL), a PTB domain construct (residues 51–238) that includes a shorter portion of the linker region (CCM2PTB-ShortLinker, CCM2PTB-SL), and a PTB domain construct (residues 51–223) predicted not to include residues of the interdomain linker (CCM2PTB; Fig. 1 A). We found that CCM3 bound to CCM2FL, CCM2PTB-LL, and CCM2PTB-SL, but displayed notably reduced binding to the PTB domain–alone construct, CCM2PTB (Fig. 1 B). Quantification revealed that CCM2PTB bound significantly lower amounts of CCM3 than the longer CCM2 constructs do (Fig. 1 C). Both CCM2PTB-LL and CCM2PTB-SL bound comparable levels of CCM3 (P = 0.478), while CCM2FL consistently bound slightly more CCM3 than CCM2PTB-LL (P = 0.01) or CCM2PTB-SL (P = 0.004). These data suggest that residues 223–238, located within the N-terminal portion of the linker between the PTB and HHD domains and containing a putative LD-like motif (Fig. 1 D), are necessary for the CCM2 interaction with CCM3.
Affiliation: Department of Pharmacology and Department of Cell Biology, Yale University, New Haven, CT 06520.