Integrins are membrane proteins with single alpha-helices transmembrane domain. Integrins
are heterodimers formed by α subunits and β subunits. More than 20 different heterodimeric integrins
are formed by 18 α subunits and 8 β subunits in mammals (Margadant, Monsuur et al. 2011). Extracellu-
lar domains of integrins are glycosylated and form functional domains to interact with ligands or other
proteins. Integrin family can be divided into two subgroups by their structures difference: with or with-
out an extra von Willebrand factor type A domain (αA or αI domain). Integrins can also be divided into
four groups based on their ligands or binding partners: collagen-binding integrins; laminin-binding
integrins; RGD dependent integrins and Leukocyte integrins. Each integrin subunit contains a bunch of
specific domains. Integrin heterodimers are illustrated as a headpiece and ‘leg’ part (Springer 2002). In-
tegrin ligand binding site is located in the headpiece.
1.4.2.1 Integrin conformation change
Integrins have two conformational structures: close and open conformation. In the close or
‘bent’ conformation, the headpiece of integrin heterodimers bends over to the ‘leg’ part. The ligand
binding site is facing to the cell membrane, which results in the low binding affinity of integrin hetero-
derstand the relationship between integrin conformation change and its function. Upon the binding of
integrin to its ligand, integrin undergoes dramatic conformational change in its extracellular domain.
This change makes integrins skewed towards clustering with adjacent integrin. The conformational
change in the extracellular domain of integrins also induces a structure change in integrin
transmembrane domain and intracellular domain (ICD), which separates integrin c-terminals of α and β
subunits. C-terminal separation of integrin α and β subunits leads to the assembly of focal adhesion
complex and activation of FAK and its further downstream signaling pathways (Askari, Buckley et al.
2009).
1.4.2.2 Integrin ligand
RGD-containing ligand is the largest protein family which binds integrins including αvβ3, α5β1,
αIIbβ3, and etc. This integrin subfamily recognizes a consensus binding sequence: three amino acids
RGD (R: Arginine, G: Glycine, D: Aspartic acid). Based on the analysis of the crystal structure, the binding
site for RGD-containing ligand locates at the interface of integrin α and β subunits (Xiong, Stehle et al.
2002). Lysine residue interacts with β-propeller domain in α subunit while aspartic acid binds to von
Willebrand factor A (vWF) domain in β subunit. Many extracellular matrix (ECM) components contain
RGD sequence including fibronectin and vitronectin. Certain ECM proteins do not have accessible RGD
sequences on the protein surface, but expose their cryptic RGD sequence to interact with RGD-specific
integrins after being denatured by temperature or cleavage by the matrix metalloproteinase
(Yamamoto, Yamato et al. 1995). For instance, Integrin α5β1 and αvβ3 are the typical RGD dependent
integrins. But collagen I does not bind to either integrin α5β1 or αvβ3 due to the absence of any solvent
accessible RGD sequence. However, after being partially denatured by increasing temperature or de-
graded by extracellular proteases (MMP-2 or MMP-9), collagen I unfolds and exposes its RGD sequence
related to cancer angiogenesis and metastasis (Eliceiri and Cheresh 1999). GRGDSP is a sequence from
fibronectin and used as an angiogenesis inhibitor as to compete with RGD-containing native ligands.
In addition, some non-ECM proteins containing RGD sequence were also reported to interact
with integrins. The human immunodeficiency virus (HIV-1) protein, transactivating factor (TAT) activates
FAK through integrin αvβ3 and promote angiogenesis (Urbinati, Mitola et al. 2005). TAT interaction with
integrin is inhibited by both RGD peptide competitive inhibitor and blocking antibody LM609, which in-
dicates that the interaction is via RGD sequence in TAT protein. Another example is CD40 ligand or
CD40L. Soluble CD40 ligand was reported to bind to integrin α5β1 and induce signaling transduction by
activating MAPK and its downstream ERK1/2 in endothelial cells (Leveille, Bouillon et al. 2007). This in-
teraction is through RGD sequence on the CD40L (Andre, Prasad et al. 2002).
Besides RGD sequence, KGD (Lysine-Glycine-Aspartic acid) is also recognized by integrins in cer-
tain cases. Integrin β3 is the major type of integrin for interacting with KGD, especially αIIbβ3. KGD plays
important role in disintegrin binding to integrin. Disintegrin is a family of proteins in viper venom.
Disintegrin inhibits platelet aggregation and causes hemorrhage through the interaction of KGD se-
quence with αIIbβ3 integrin (Lu, Lu et al. 2005).
A synergetic site might be important for integrin binding to RGD. RGD peptides are used for can-
cer therapy to target cancer angiogenesis. RGD peptide such as cilengitide a cyclic RGD pentapeptide has
been applied as an angiogenesis inhibitor. However, the affinity of RGD peptide is 1000 fold lower than
their natural ligand. One of the most important reason is that integrin ligand contains not only RGD se-
quence but also synergetic site. Pro–His–Ser–Arg–Asn (PHSRN) is a synergetic binding site in fibronectin
for integrin α5β1 and this synergy site facilitates HUVEC cell attachment and spreading in vitro
(Ochsenhirt, Kokkoli et al. 2006).
Other than RGD sequence, LDV is another major binding sequence for integrins. LDV mainly
such as IDA or REDV (Mould and Humphries 1991). As mentioned earlier, a subgroup of integrins contain
α domain, α1β1, α2β1, α10β1 and α11β1. Those integrin binds to specific residues other than RGD and
LDV. GFOGER is the critical sequence on collagen to be recognized by αA-containing integrins. The glu-
tamic acid residue in the recognition sequence is crucial to interact with the metal in the integrins
(Emsley, Knight et al. 2000).
1.4.2.3 Metal binding vs. Ligand binding
Magnesium and calcium metal ions are crucial to maintain integrin proper structure and ligand
binding activity. As described above, integrin can be divided into two subfamilies based on their struc-
ture similarity: αA integrins and non-αA integrins. αA integrins contain a von Willebrand factor type A
domain. αA is responsible for mediating integrins binding to their ligands and α domain contains an ion
binding site, named metal-ion-dependent adhesion site (MIDAS) (Michishita, Videm et al. 1993). MIDAS
coordinates one divalent cation, usually Magnesium and MIDAS domain accepts one interaction from an
acidic amino acid from integrin ligand (Lee, Rieu et al. 1995). MIDAS has even higher binding affinity for
Manganese (Li, Rieu et al. 1998). However, Manganese is not a physiological ion. Although structure dif-
ferent exists between αA-containing and α-lacking Integrins, integrins without αA domain shows similar
strategy for ligand binding. Integrin αvβ3 is an important αA-lacking integrin. αvβ3 contains an αA-like
domain or βA/βI domain (Fig 1-5)(Luo and Springer 2006). Aspartic acid in RGD tripeptide attaches to
the ion located at MIDAS site, while Arginine (R) residue interacts with a couple of negative charged res-
idues at β propeller on αsubunit. MIDAS on integrin β subunit and β propeller domain from α subunit
forms the basic ligand binding pocket. Besides MIDAS at αA domain or A-like domain on integrin β subu-
nit, other two metal binding sites locates adjacent to MIDAS: the ADMIDAS (adjacent to MIDAS) and
LIMBS (ligand-associated metal binding site). Those sites favor calcium ion and provide additional regu-
transmembrane protein, which highly expresses in tumor endothelium.TEM8 also contains an MIDAS
site and this MIDAS was reported to be involved in binding to the anthrax toxin antigen (Ramey, Villareal
et al. 2010). The interaction of TEM8 and its ligand is much similar to the binding of integrin and RGD
peptide. It was reported that PKM2 also could bind to TEM8 in vitro (Duan, Hu et al. 2007). This interac-
tion indicates the possibility that PKM2 also binds to MIDAS on the integrin.
Integrin α subunit also contains 5 Calcium binding sites. 4 of them are in beta propeller loop re-
gion and the other one is at ‘genu’ region, which acts as a ‘knee’ for integrin conformational change
(Arnaout, Goodman et al. 2007). Metal ions are required for integrin structure and integrin ligand bind-
ing. EDTA completely inhibits integrin function and detaches cells.