Changing Ligand Specificities of αvβ1 and αvβ3 Integrins by Swapping a Short Diverse Sequence of the β Subunit (original) (raw)
Related papers
Integrin αIIbβ3: ligand interactions are linked to binding‐site remodeling
2006
This studyt ested theh ypothesis that high-affinity bindingo fm acromolecularl igands to the a IIbb 3 integrin is tightlyc oupled to binding-site remodeling,a ni nduced-fit processt hats hiftsaconformational equilibriumf romaresting toward an open receptor.I nteractionsb e tween a IIb b 3a nd twom odel ligands-echistatin,a6-kDarecombinant proteinwithanRGD integrin-targeting sequence,and fibrinogen's g -module, a3 0-kDar ecombinant proteinw ith aK QAGDVi ntegrin bindings ite-were measured by sedimentationvelocity, fluorescence anisotropy,a nd as olid-phase bindinga ssay,a nd modeledbymolecular graphics.Studyingechistatinvariants(R24A,R24K, D26A,D26E, D27W,D27F),wefound that electrostatic contacts with chargedr esiduesa tt he a IIb/b 3i nterface,r athert hann onpolarc ontacts, perturbt he conformation of ther esting integrin.A spartate 26,w hich interacts with then earbyM IDAS cation,w as essentialfor binding, as D26A andD26Ewereinactive. In contrast,R24Kwas fullyand R24A partly active, indicating that thepositivelycharged arginine 24 contributesto, butisnot required for, integrin recognition. Moreover,w ed emonstrated that priming-i.e.,e ctodomainc onformationalc hanges ando ligomerization inducedbyincubationat35°Cwiththe ligand-mimetic peptidecHarGD-promotes complexformation with fibrinogen's g -module. We also observed that the g -module'sflexiblecarboxy terminus wasnot required for a IIb b 3i ntegrinb inding.O ur studiesd ifferentiate primingl igands,w hich bind to ther esting receptor and perturbits conformation,fromregulated ligands, wherebinding-siteremodelingmustfirst occur. Echistatin's bindingenergyissufficienttorearrange thesubunit interface, butregulated ligandslikefibrinogenmustrely on primingt oo vercomec onformational barriers.
Multiple Binding Sites in Fibrinogen for Integrin αMβ2 (Mac-1)
Journal of Biological Chemistry, 2004
The leukocyte integrin ␣ M  2 (Mac-1) is a multiligand receptor that mediates a range of adhesive reactions of leukocytes during the inflammatory response. This integrin binds the coagulation protein fibrinogen providing a key link between thrombosis and inflammation. However, the mechanism by which ␣ M  2 binds fibrinogen remains unknown. Previous studies indicated that a model in which two fibrinogen ␥C domain sequences, P1 (␥190-202) and P2 (␥377-395), serve as the ␣ M  2 binding sites cannot fully account for recognition of fibrinogen by integrin. Here, using surface plasmon resonance, we examined the interaction of the ligand binding ␣ M I-domain of ␣ M  2 with the D fragment of fibrinogen and showed that this ligand is capable of associating with several ␣ M I-domain molecules. To localize the alternative ␣ M I-domain binding sites, we screened peptide libraries covering the complete sequences of the ␥C and C domains, comprising the majority of the D fragment structure, for ␣ M I-domain binding. In addition to the P2 and P1 peptides, the ␣ M I-domain bound to many other sequences in the ␥C and C scans. Similar to P1 and P2, synthetic peptides derived from ␥C and C were efficient inhibitors of ␣ M  2-mediated cell adhesion and were able to directly support adhesion suggesting that they contain identical recognition information. Analyses of recognition specificity using substitutional peptide libraries demonstrated that the ␣ M I-domain binding depends on basic and hydrophobic residues. These findings establish a new model of ␣ M  2 binding in which the ␣ M I-domain interacts with multiple sites in fibrinogen and has the potential to recognize numerous sequences. This paradigm may have implications for mechanisms of promiscuity in ligand binding exhibited by integrin ␣ M  2 .
Specific Binding of Integrin αvβ3 to the Fibrinogen γ and αE Chain C-Terminal Domains
Biochemistry, 1999
Integrin Rv 3, a widely distributed fibrinogen receptor, recognizes the RGD 572-574 motif in the R chain of human fibrinogen. However, this motif is not conserved in other species, nor is it required for Rv 3-mediated fibrin clot retraction, suggesting that fibrinogen may have other Rv 3 binding sites. Fibrinogen has conserved C-terminal domains in its R (E variant), , and γ chains (designated R E C, C, and γC, respectively), but their function in cell adhesion is not known, except that RIIb 3, a platelet fibrinogen receptor, binds to the γC HHLGGAKQAGDV 400-411 sequence. Here we used mammalian cells expressing recombinant Rv 3 to show that recombinant R E C and γC domains expressed in bacteria specifically bind to Rv 3. Interaction between Rv 3 and γC or R E C is blocked by LM609, a functionblocking anti-Rv 3 mAb, and by RGD peptides. Rv 3 does not require the HHLGGAKQAGDV 400-411 sequence of γC for binding, and R E C does not have such a sequence, indicating that the Rv 3 binding sites are distinct from those of RIIb 3. A small fragment of γC (residues 148-226) supports Rv 3 adhesion, suggesting that an Rv 3 binding site is located within the γ chain 148-226 region. We have reported that the CYDMKTTC sequence of 3 is responsible for the ligand specificity of Rv 3. γC and R E C do not bind to wild-type Rv 1, but do bind to the Rv 1 mutant (Rv 1-3-1), in which the CYDMKTTC sequence of 3 is substituted for the corresponding 1 sequence CTSEQNC. This suggests that γC and R E C contain determinants for fibrinogen's specificity to Rv 3. These results suggest that fibrinogen has potentially significant novel Rv 3 binding sites in γC and R E C.
The role of the I domain in ligand binding of the human integrin alpha 1 beta 1
Journal of Biological Chemistry, 1994
We report here the analysis of potential ligand binding domains within the human integrin a1 subunit, a known collagedaminin receptor. This integrin is effectively blocked by the mouse monoclonal antibody 1B3.1. A truncated version of the a1 subunit lacking the NH,terminal half of the extracellular domain is not recognized by monoclonal antibody 1B3.1. Furthermore, we have isolated a cDNA containing the I domain from chicken a1 bearing significant homology to the human and rat a1 sequences. Replacing the human I domain with its chicken counterpart led to the surface expression of a functional heterodimer with endogenous mouse p, on NIH 3T3 cells. However, 1B3.1 does not bind to the chickenhuman chimera, demonstrating that the human al I domain is required for epitope recognition. Mutation of Aspzs3 within the I domain to alanine resulted in surface expression of an ap heterodimer recognized by 1B3.1 but with markedly reduced binding to collagen N or laminin. Since a previously reported mutation of a homologous Asp in the Mac-1 I domain has similar consequences, these results suggest a central role for the I domain in ligand recognition for all integrin a subunits containing this domain. Integrins, a family of cell surface receptors, have a prominent role in cell-cell and cell-matrix adhesion events important for processes such as embryonic development and morphogenesis, hemostasis, and thrombosis, wound healing, metastasis, and leukocyte helper and killer function. Integrins recognize a wide variety of ligands including extracellular matrix proteins, serum proteins, and counter-receptors on other cells. Integrins are expressed as heterodimers composed of noncovalently associated a and p subunits. At least 15 distinct a and 8 distinct p subunits have been identified and the dimers formed between them are divided into classes based on their subunit (1). The a subunits, which have a repeating modular structure, can be separated into two groups, with the exception of aq (2). The first type of a subunit has a post-translational intrachain cleavage and four discrete metal binding domains, ~ ~~ GM 44585 and by the Council for Tobacco Research, Inc. Grant 2906. * This work was supported by National Institutes of Health Grant The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBankmlEMBL Data Bank with accession number(s) U10114. $ Recipient of a long term fellowship from The Human Frontier Science Organization no. LT 653. ll Recipient of a junior faculty award from the American Cancer So-630 w. 168th St., Black Bldg.
The structural bases of integrin-ligand interactions
Trends in Cell Biology, 1994
noncovalently associated u and [~ subunits. At least bases ul-ct and 8 13 subunits have been identified. On the basis of the subunits [3~, [32 and [33, the integrins were originally divided into three subfamilies: the [3~ and integrin ligand [33 subfamilies are inv°lved primarily in interacti°ns with ECM proteins, whereas the 132 subfamily, which . . is restricted to leukocytes, is also important for interactions cell-cell contact. The [31 subunit can combine ,,ith 10 et subunits (ot~ to ct 9, and cx~) to form heterodimers that specifically bind to ECM constituents such as fibronectin, laminin and different collagen types. The two major integrins of the [3 3 subfamily, U.b[33 and av[33, are mainly directed against vascular li-Many extracellular matrix (ECM) proteins, particularly those in gands, such as fibronectin, vitronectin, fibrinogen, von Will brand factor, thrombospondin and others the vascular system, use their classical integrin-recognition motif (Table 1).
A Molecular Basis for Integrin αMβ2 Ligand Binding Promiscuity
Journal of Biological Chemistry, 2002
The leukocyte integrin ␣ M  2 is a highly promiscuous leukocyte receptor capable of binding a multitude of unrelated ligands. To understand the molecular basis for the broad ligand recognition of ␣ M  2 , the inter-integrin chimera was created. In the chimeric integrin, the D-␣5 loop-␣5 helix segment comprised of residues Lys 245-Arg 261 from the ␣ M I domain of ␣ M  2 was inserted into the framework of ␣ L  2. The construct was expressed in HEK 293 cells, and the ability of generated cells to adhere to fibrinogen and its derivatives was characterized first. Grafting the ␣ M (Lys 245-Arg 261) sequence converted ␣ L  2 into a fibrinogen-binding protein capable of mediating efficient and specific adhesion similar to that of wild-type ␣ M  2. Verifying a switch in the binding specificity of ␣ L  2 , the chimeric receptor became competent to support cell migration to fibrinogen. Mutations at positions Phe 246 , Asp 254 , and Pro 257 within Lys 245-Arg 261 of ␣ M  2 produced significant decreases in cell adhesion, illustrating the critical role of these residues in ligand binding. The insertion of ␣ M (Lys 245-Arg 261) imparted to the chimeric integrin the ability to recognize many typical ␣ M  2 protein ligands. Furthermore, cells expressing the chimeric receptor, but not ␣ L  2 , were able to stick to uncoated plastic, which represents the hallmark of wild-type ␣ M  2. These results suggest that ␣ M (Lys 245-Arg 261) serves as a consensus binding site for interaction with a variety of distinct molecules and, thus, may define the degenerate recognition properties inherent to ␣ M  2 .
The EMBO Journal, 1995
Integrin a401 is a receptor for vascular cell adhesion molecule (VCAM)-1 and fibronectin (CS-1). The a401ligand interaction is involved in the pathogenesis of diseases and is, therefore, a therapeutic target. Here, we identified critical residues of a4 for ligand binding using alanine-scanning mutagenesis of the previously localized putative ligand binding sites (residues 108-268). Among 43 mutations tested, mutations of Tyrl87, Trpl88 and Glyl90 significantly inhibited cell adhesion to both VCAM-1 and CS-1. This inhibition was not due to any gross structural changes of a44p1. These critical residues are clustered in a predicted ,3-turn structure (residues 181-190) of the third N-terminal repeat in a4. The repeat does not contain divalent cation binding motifs. Notably, the mutations within the corresponding region of aS significantly reduced fibronectin-a5jx1 interaction. These findings suggest that the predicted 13-turn structure could be ubiquitously involved in ligand binding of non-I domain integrins.