Amino Acid Residues in the αIIb Subunit That Are Critical for Ligand Binding to Integrin αIIbβ3 Are Clustered in the β-Propeller Model (original) (raw)

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.

Binding of a fibrinogen mimetic stabilizes integrin αIIbβ3's open conformation

Protein Science, 2001

The platelet integrin ␣IIb␤3 is representative of a class of heterodimeric receptors that upon activation bind extracellular macromolecular ligands and form signaling clusters. This study examined how occupancy of ␣IIb␤3's fibrinogen binding site affected the receptor's solution structure and stability. Eptifibatide, an integrin antagonist developed to treat cardiovascular disease, served as a high-affinity, monovalent model ligand with fibrinogen-like selectivity for ␣IIb␤3. Eptifibatide binding promptly and reversibly perturbed the conformation of the ␣IIb␤3 complex. Ligand-specific decreases in its diffusion and sedimentation coefficient were observed at near-stoichiometric eptifibatide concentrations, in contrast to the receptorperturbing effects of RGD ligands that we previously observed only at a 70-fold molar excess. Eptifibatide promoted ␣IIb␤3 dimerization 10-fold more effectively than less selective RGD ligands, as determined by sedimentation equilibrium. Eptifibatide-bound integrin receptors displayed an ectodomain separation and enhanced assembly of dimers and larger oligomers linked through their stalk regions, as seen by transmission electron microscopy. Ligation with eptifibatide protected ␣IIb␤3 from SDS-induced subunit dissociation, an effect on electrophoretic mobility not seen with RGD ligands. Despite its distinct cleft, the open conformer resisted guanidine unfolding as effectively as the ligand-free integrin. Thus, we provide the first demonstration that binding a monovalent ligand to ␣IIb␤3's extracellular fibrinogen-recognition site stabilizes the receptor's open conformation and enhances self-association through its distant transmembrane and/or cytoplasmic domains. By showing how eptifibatide and RGD peptides, ligands with distinct binding sites, each affects ␣IIb␤3's conformation, our findings provide new mechanistic insights into ligand-linked integrin activation, clustering and signaling.

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 α_E 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.

Multiple Binding Sites in Fibrinogen for Integrin M 2 (Mac-1)

Journal of Biological Chemistry, 2004

Changing Ligand Specificities of αvβ1 and αvβ3 Integrins by Swapping a Short Diverse Sequence of the β Subunit

Journal of Biological Chemistry, 1997

Integrins mediate signal transduction through interaction with multiple cellular or extracellular matrix ligands. Integrin ␣v␤3 recognizes fibrinogen, von Willebrand factor, and vitronectin, while ␣v␤1 does not. We studied the mechanisms for defining ligand specificity of these integrins by swapping the highly diverse sequences in the I domain-like structure of the ␤1 and ␤3 subunits. When the sequence CTSEQNC (residues 187-193) of ␤1 is replaced with the corresponding CYD-MKTTC sequence of ␤3, the ligand specificity of ␣v␤1 is altered. The mutant (␣v␤1-3-1), like ␣v␤3, recognizes fibrinogen, von Willebrand factor, and vitronectin (a gain-of-function effect). The ␣v␤1-3-1 mutant is recruited to focal contacts on fibrinogen and vitronectin, suggesting that the mutant transduces intracellular signals on adhesion. The reciprocal ␤3-1-3 mutation blocks binding of ␣v␤3 to these multiple ligands and to LM609, a function-blocking anti-␣v␤3 antibody. These results suggest that the highly divergent sequence is a key determinant of integrin ligand specificity. Also, the data support a recent hypothetical model of the I domain of ␤, in which the sequence is located in the ligand binding site.

Amino Acid Residues in the αIIb Subunit That Are Critical for Ligand Binding to Integrin αIIbβ3 Are Clustered in the β-Propeller Model (original) (raw)

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