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Padarti et al. Vessel Plus 2018;2:21 I http://dx.doi.org/10.20517/2574-1209.2018.34 Page 5 of 23

After some invertebrate analogs of CCM3 were identified and studied, it was found that CCM3 is the most
evolutionarily conserved of the three CCM proteins [64,65] . However, CCM1 and CCM2 are predominantly
found in the vertebrates [63,66] .

CCM3 N-terminal dimerization domain
CCM3 exists as a homodimer in the cell due to presence of the dimerization domain. It is made up of four
α-helices: α1, α2, α3, and small α4. These four α-helices interlock with another set of four α-helices from
a partner CCM3 dimerization domain. This dimerization domain is also used to bind to GCKIII kinases
forming a heterodimer. The interactions between GCKIII and flexible hinge regions of CCM3 control the
equilibrium between CCM3 homodimer and CCM3-GCKIII heterodimer .
[45]
CCM3 C-terminal focal adhesion targeting - homology domain
The C-terminus of the CCM3 protein contains a focal adhesion targeting-homology domain (FAT-H) domain.
This domain is commonly found in some tyrosine kinases such as Pyk2 and FAK. It is also composed of four
α-helices: α5, α6, α7, and α8. This domain is utilized to bind to large variety proteins including CCM2 and
phosphotidylinositides .
[62]

NEWLY IDENTIFIED CELLULAR COMPONENTS OF CSC COMPLEX
CCM1 is highly expressed in EC cells during embryogenesis . It is localized ubiquitously throughout the
[67]
cell including the nucleus [17,20,38,42,68] .

CCM1 C-terminal FERM domain
CCM1 was shown to bind to a small GTPase Rap1 utilizing its FERM domain through X-ray
crystallography [43,44] and yeast two-hybrid . This interaction localizes CCM1 to the periphery of the cell to
[69]
facilitate signaling at the cell-cell junctions . In contrary, the release of CCM1 from the plasma membrane
[48]
is due to its interaction with ICAP1α. This complex localizes to the nucleus . Rap1 is a GTPase that has
[17]
many cellular functions such as maintaining cell-cell contacts and integrin-mediated cell adhesion . Rap1
[70]
is activated by GTPase-activating proteins and CCM1 has a large affinity towards this activated form .
[71]
Disruption of RAP1 and CCM1 interaction results in the absence of CCM1 localization to the cell membrane
particularly the adherens junction . Superimposition of Rap1 and CCM1 showed that no conformational
[71]
change was required for bindings to occur . The FERM domain contains 3 subdomains F1, F2, and F3.
[52]
RAP1 binding site in the FERM domain overlaps F1 and F2 subdomains. HRas is another GTPase that binds
to the same binding pocket as RAP1. However, in competition assays, RAP1 binds more strongly to CCM1
over HRas due to specific residues in the F2 subdomain of CCM1. This stronger binding affinity to RAP1 is
dependent on the interaction of K570 on CCM1 with E45 on RAP1, with respective mutations significantly
decreasing binding. Further analysis of the X-ray crystallography of CCM1 and RAP1 showed that the
switch II region in RAP1 doesn’t contribute to any binding affinity. This is a result of the interaction between
Y419 on CCM1 and F64 on RAP1 which destabilizes the switch II region . Rap1 acts as an inhibitor of
[43]
CCM1 interaction with microtubules . The inhibition mechanism is the binding of Rap1, releasing CCM1
[48]
from the cell membrane and spatially blocking the interaction between CCM1 and microtubules leading to
an overall increase in stability of the cell junction . Rap1 is found in mice in two forms: Rap1a and Rap1b.
[72]
Individual deletions of either protein have minimal effect on embryogenesis. However, a combined deletion
is embryo lethal. It results in the perineural vessel dilation and hemorrhage, demonstrating the importance
of Rap1 in EC cells maturation and angiogenesis. Rap1 deletion was shown to reduce VEGFR2 signaling .
[73]
Unlike CCM1 deletions which result in malformed branchial arches, the formation of patent branchial
arches can still be seen in Rap1 deletion, suggesting that CCM1 acts along alternate pathways for vessel
formation . CCM1 FERM domain also binds to HEG1 protein, which further enables CCM1 localization
[74]
at the cell junction. HEG1 is a transmembrane protein that contains an NPxY motif on its cytoplasmic
tail. The binding pocket of HEG1 in CCM1 does not overlap with RAP1. The binding strength of HEG1 to