The Eukaryotic Linear Motif resource for
Functional Sites in Proteins
Functional site class:
Integrin RGD-type binding sites
Functional site description:
Integrins are cell adhesion-mediating receptors present in all metazoans. Each integrin is composed of one α and one β subunit; in humans, 18 α and 8 β subunits can combine to form 24 different dimers, each with unique ligand specificities. Eight of the human integrin dimers can recognize ligands with RGD motifs [D'Souza,1991], present in several proteins from humans and pathogens or parasites. The RGD core motif fits into a deep groove between the two subunits with the Arg residue contacting the α subunit and the Asp residue coordinating a divalent cation embedded in the β subunit, held in place by the Metal-Ion-Dependent Adhesion Site (MIDAS) [Xiong,2002], while the flanking residues modify specificity and affinity. The Arg can be replaced by other residues in certain ligands. In addition, an NGR sequence region can naturally degrade into isoDGR (where isoD is an L-Asp residue) through spontaneous deamidation, creating a functional reverse RGD-like binding motif [Curnis,2006].
ELMs with same func. site: LIG_Integrin_isoDGR_2  LIG_Integrin_KxxGD_FGGC_5  LIG_Integrin_RGD_1  LIG_Integrin_RGDSP_6  LIG_Integrin_RGD_TGFB_3  LIG_Integrin_RGDW_4 
ELM Description:
NGR (asparagine-glycine-arginine) is a tripeptide present in fibronectin (FINC_HUMAN), fibrillin-1 (FBN1_HUMAN), and adeno-associated virus 2 protein capsid (CAPSD_AAV2S). Asparagine deamidation of NGR motif yields isoDGR (isoaspartic acid-glycine-arginine) motif. Deamidation is a non-enzymatic process that involves formation of succinimide intermediate and subsequent formation of isoDGR by hydrolysis. The isoDGR motif is able to interact with integrins by recognition of RGD-binding site of several integrins including αVβ3 integrin. RGD composite binding site is formed by both alpha and beta subunits of integrins and is located on the extracellular side of plasma membrane. IsoDGR binds to RGD-binding site in inverted orientation compared to proteins that contain RGD motif (Spitaleri,2008). CisoDGRC is a cyclic peptide that competes with RGD-containing peptides for binding to αVβ3 integrins (Curnis,2006). CisoDGRC binding to RGD-binding pocket inhibits endothelial cell adhesion, proliferation, and tumour growth. Both ligands have similar binding affinity for αVβ3 integrin. CisoDGRC binds to the following integrins with decrease in affinity: αVβ3, α5β1, αVβ6, αVβ5, αVβ8. Linear isoDGR (GisoDGRG) binds to αVβ6, αVβ3, α5β1, αVβ5, and αVβ8. Cyclic isoDGR binds with 10-100-fold increased affinity to αVβ3 compared to other integrins. Acetylation of either linear or cyclic isoDGR increases the affinity for integrins accompanied by loss of specificity. Peptide linearization (replacing flanking glycines with cysteines) is associated with 100-fold loss of αVβ3 binding affinity and specificity. Hence, flanking residues of NGR motif affect affinity and specificity for integrin binding (Curnis,2010).
Pattern: NGR
Pattern Probability: 0.0001597
Present in taxons: Bos taurus Canis lupus familiaris Danio rerio Gallus gallus Homo sapiens Metazoa Pan troglodytes Rattus norvegicus Sus scrofa Xenopus laevis
Interaction Domains:
o See 8 Instances for LIG_Integrin_isoDGR_2
o Abstract
Integrins are metazoan-specific receptors not present in the other crown group eukaryotes fungi or viridiplantae. All human cells express one or more of the 24 types of dimeric integrins spanning the plasma membrane [Barczyk,2009], which mediate signals between the intracellular space, and neighbouring cells or the extracellular matrix [Takada,2007; Campbell,2011; Hynes,2002]. The presence and ratio of various integrins reflect the cell’s function. Eight of the human integrins (αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, α5β1, α8β1 and αIIbβ3), which resemble the evolutionarily most ancient metazoan integrins, can recognize RGD and RGD-like sequence patterns in their ligands [D'Souza,1991]: components of the extracellular matrix (ECM), cell surface proteins of cells or other extracellular signaling proteins. These interactions are central to regulating tissue integrity and tissue boundary formation [Julich,2015], blood clotting [Hook,2017], angiogenesis [Atkinson,2014] and bone formation [Marie,2014], and regulating nutrient absorption through gastrointestinal motility [Khalifeh-Soltani,2016], amongst other functions.

Due to their central roles in cellular communication, misregulation of integrins is implicated in a wide range of diseases. Several viruses, such as the foot-and-mouth disease virus, HIV, West Nile or HPV-16 [Hussein,2015; Asokan,2006] have RGD-like motifs embedded in their proteins that can attach to integrins on the host cell surface, aiding cell entry. Several other pathogens, including both bacteria and eukaryotes also harbour RGD-like motifs to interface with the host cells. Integrins are also known to be targeted by disintegrins [Calvete,2003], a class of proteins present in venoms of snakes from the Viperidae family, ticks, leeches and other parasites. Disintegrins form the strongest known integrin interactions with typical affinities in the low nanomolar - high picomolar range. In addition to pathogenesis, endogenous integrin misregulation is connected to non-pathogenic conditions including Alzheimer’s [Donner,2016], cystic fibrosis [Reed,2015], autism spectrum disorder and schizophrenia [Lilja,2018]. A focal point of therapeutic integrin research is cancer [Seguin,2015], as integrins play pivotal roles in angiogenesis and metastasis. Yet, despite the nearly 80,000 publications on integrins, only a handful of integrin-drugs are available commercially, all targeting RGD-binding integrins. Eptifibatide (an antithrombotic drug), which is a result of semi-rational peptide design, is the only one where the integrin interacting region of a snake venom disintegrin was successfully copied and integrated into a cyclic peptide [Phillips,1997]. Apart from Eptifibatide, such efforts have also produced promising anti-cancer drug candidates, such as Cilengitide and peptides developed to slow down neovasculature formation [Corti,2008]. Other preliminary results show that integrin antagonists could provide a means against inflammatory diseases [Maiguel,2011], HIV infection [Arthos,2018], or could be used in regenerative medicine [Rocha,2018].

One of the reasons for the complexity of integrin regulation is the sensitivity of the downstream signaling to the structural details of ligand binding. While the core RGD motif is common to a wide range of ligands, the exact structural details of the binding determine if the ligand acts as a full or partial agonist or antagonist. There are four major alterations/additions to the presence of the RGD motif that influence this agonistic/antagonistic behaviour, as well as tuning the affinity of the binding and the selectivity profile of the ligand (i. e. which integrin dimers can it bind to):
- First, the flanking residues of the core RGD motif, especially the residues following the Asp, have a huge influence on selectivity and binding strength. Certain integrins have multiple binding modes and these flanking residues are able to determine which binding mode a given ligand will use. For example αv αvβ6 and αvβ8 integrins can bind ligands where the RGD and the following sequence region are in coil conformation, such as fibronectin. However, the same integrins can also bind ligands where RGD is followed by a short helix interacting with the β6 or β8 subunit via hydrophobic contacts, such as for TGFβ-1 and -3. The two binding modes require different C-terminal flanking residues and influence the binding strength to the same integrins.
- Second, the Arg residue in RGD can be replaced with other residues, most notably Lys, and it can also have a variable position taking advantage of the different side chain length of Lys compared to Arg. Since the interactions formed by Asp itself can be sufficient for biologically relevant binding, the positive charge of RGD can even be omitted in some functional motif instances.
- Third, integrins can bind their ligands in an inverted orientation using a reverse motif. In this case, the Asp residue has to be replaced by its mirror image pair, namely L-Asp. Under physiological conditions, Asn residues followed by Gly can spontaneously decay into L-Asp via spontaneous deamidation [Corti,2011; Curnis,2010]. Hence, NGR sequence regions can transform into isoDGR (where isoD represents L-Asp) and they can be actively converted back to NGR by the enzyme protein-L-isoaspartate (D-aspartate) O-methyltransferase (P22061). Natural ligands harbour either an RGD or an NGR motif and some ligands, such as fibronectin, contain both [Curnis,2006].
- Fourth, functional RGD-like motifs often occur in both disordered and ordered regions of proteins. This is in contrast with the notion that most functional short linear motifs reside in disordered protein segments as they need to structurally adapt to their binding partner. However, RGD-like motifs need to adopt a β-turn like conformation to fit into the binding pocket of integrins, and extended surface loops of ordered domains can effectively mimic this conformation. The ordered/disordered nature of an RGD-like motif can heavily influence its binding affinity. As an ordered motif does not lose much conformational entropy upon binding, RGD motifs achieving extremely low Kd values (such as disintegrins [Arruda Macedo,2015]) are most often part of ordered structure, leading to non-transient binding. In contrast, intrinsically flexible ligands such as osteopontin or nephronectin are often disordered to enable a more transient and reversible interaction.
o 10 selected references:

o 7 GO-Terms:

o 8 Instances for LIG_Integrin_isoDGR_2
(click table headers for sorting; Notes column: =Number of Switches, =Number of Interactions)
Acc., Gene-, NameStartEndSubsequenceLogic#Ev.OrganismNotes
P35555 FBN1
2304 2306 QTKPGICENGRCLNTRGSYT TP 4 Homo sapiens (Human)
P11276 Fn1
501 503 MMRCTCVGNGRGEWACIPYS TP 3 Mus musculus (House mouse)
P11276 Fn1
264 266 LLQCVCTGNGRGEWKCERHA TP 2 Mus musculus (House mouse)
P02751 FN1
1432 1434 YVVSIVALNGREESPLLIGQ TP 4 Homo sapiens (Human)
P02751 FN1
501 503 MMRCTCVGNGRGEWTCIAYS TP 5 Homo sapiens (Human)
P02751 FN1
367 369 PCVLPFTYNGRTFYSCTTEG TP 4 Homo sapiens (Human)
P02751 FN1
263 265 LLQCICTGNGRGEWKCERHT TP 6 Homo sapiens (Human)
P03135 Capsid protein VP1
511 513 TGATKYHLNGRDSLVNPGPA TP 6 Adeno-associated virus 2 Srivastava/1982
Please cite: The Eukaryotic Linear Motif resource: 2022 release. (PMID:34718738)

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