Integrins are transmembrane receptors responsible primarily for cell migration and extracellular matrix adhesion. Integrins are heterodimers, composed of one alpha, and one beta subunit. They function through bidirectional signalling. There are 18 alpha and 8 beta subunits in the integrin family that assemble into 24 heterodimers. alpha subunits determine integrin ligand specificity whereas beta subunits are connected to the cytoskeleton. alpha and beta subunits are held by non-covalent interactions. Integrins are able to bind a variety of ligands including cell surface adhesion proteins and extracellular matrix proteins. Integrin signalling involves assembly of receptor-ligand complexes on extracellular side of plasma membrane. Integrin proteins are present only in metazoa with no integrins found in fungi, plants, or prokaryotes. The structure of integrin includes extracellular domain which contains ligand binging sites, plasma membrane regions, and short cytoplasmic domains (19693543, 21421922).
A hallmark of integrins is the ability of individual family members to recognize multiple ligands. Most integrins recognize relatively short peptide motifs such as RGD, LDV, DLXXL, NGR and, in general, a key constituent residue is an acidic amino acid. Some collagens have another - different - integrin binding motif. The ligand specificities rely on both subunits of a given α-β heterodimer. Proteins that contain RGD motifs recognise 8 out of 24 integrins. RGD was originally identified as the sequence in fibronectin that engages the fibronectin receptor, integrin α5β1. RGD sequences have also been found to be responsible for the cell adhesive properties of a number of other proteins, including fibrinogen, victronectin, von Willebrand factor and many other glycoproteins. Many snake venoms are rich in RGD peptides - a testament to the importance of the integrin system. While their motifs may be more benign, the pharmaceutical industry also finds the integrin-RGD system to be of considerable interest. Antagonists could be effective for therapeutic intervention in cancer, thrombosis and numerous inflammatory conditions.
The IsoDGR motif of integrin-binding ligands arises from the NGR motif via deamidation of asparagine (N) or from the DGR motif via isomerisation of aspartic acid (D). Deamidation is a non-enzymatic reaction that involves the formation of succinimide intermediate and its hydrolysis generating aspartic acid and isoaspartic acid (isoD) residues. The ratio of aspartic acid to isoaspartic acid residues during the deamidation process is 1:3. The D-isoDGR peptide was shown to poorly compete for the RGD binding site. Hence the interaction of isoDGR with integrin is stereospecific favouring L-isomer with L-isoaspartic acid residues being most predominant after deamidation (17015452, 21282473). One of the factors that control the rate of deamidation is the presence of specific amino acids near the asparagine or isoaspartic acid residues. For example glycine near these residues accelerates the process of isoDGR formation (20064928). Newly formed isoDGR motif mimics a RGD motif and recognises the RGD-binding site of integrins (21282473). One of the proteins that binds to certain integrins via an isoDGR motif is fibronectin (FN). Fibronectins are proteins involved in cell adhesion, motility, shape maintenance, and healing. There are two highly conserved NGR sequences in orthologues of fibronectin. 5th and 7th FN-I repeats each contain a NGR motif that is conserved in human, rat, bird, murine, amphibian, and fish proteins. Both NGR sequences are flanked by glycine residues (GNGRG). IsoDGR motif of fibronectin interacts with integrins by recognition of RGD-binding site of αVβ3, αVβ5, αVβ6, αVβ8, and α5β1 integrins. Integrins such as α1β1, α3β1, α4β7, α5β7, α6β4, and α9β1 cannot be recognised by isoDGR-containing peptides (17015452). Full-length plasma fibronectin is resistant to asparagine deamidation compared to short fibronectin fragments or peptides containing NGR motif. Hence deamidation of NGR in fibronectin requires proteolytic cleavage. Intracellular enzyme protein-L-isoAsp-O-methyltransferase (PIMT_HUMAN) can inhibit the effects of asparagine deamidation. PIMT converts L-isoaspartic acid and D-aspartic acid to L-aspartic acid residues through methyl-esterification. PIMT can only target extracellular proteins when it is released into extracellular space by damaged vessels and injured tissues. PIMT restores the primary amino acid sequence in case of aspartic acid isomerisation but not in case of asparagine deamidation (18574027, 21282473).
NGR and isoDGR motifs might have therapeutic applications that are currently being evaluated. IsoDGR motif-containing peptides are able to recognise αVβ3 integrin-positive endothelial cells in tumour vessels and inhibit tumour growth in tumour-bearing mice as well as inhibit endothelial cells proliferation and adhesion to vitronectin. IsoDGR peptides can be exploited as integrin antagonists for the treatment of cancer and other diseases since isoDGR competes with RGD-containing protein for the RGD-binding pocket (21282473). NGR motif can recognise tumour vasculature by binding to aminopeptidase N (CD13). CD13 is a membrane-bound metalloproteinase that has been implicated in tumour angiogenesis. It is barely expressed by endothelium of normal blood vessels but is significantly upregulated in angiogenic tumour blood vessels. NGR peptide was also shown to target CD13 in inflammation and retinal disorders. Cyclic NGR (NGR flanked by single cysteine on both sides) peptide binds to CD13-positive blood vessels in tumours but not to epithelium of normal kidney or other CD13-rich tissues. First anticancer drug that was coupled to NGR peptide was doxorubicin. It showed reduced toxicity and improved efficacy against human cancer xenografts in nude mice compared to free doxorubicin. NGR peptide has also been coupled to tumour necrosis factor α (TNF-α) that has improved anti-tumour activity. This compound has underwent phase I and phase II trials with 50% of patients treated being stabilised, and having limited toxicity (18574027, 21282473).