RESUMEN
Although several members of protein disulfide isomerase (PDI) family support thrombosis, other PDI family members with the CXYC motif remain uninvestigated. ERp46 has 3 CGHC redox-active sites and a radically different molecular architecture than other PDIs. Expression of ERp46 on the platelet surface increased with thrombin stimulation. An anti-ERp46 antibody inhibited platelet aggregation, adenosine triphosphate (ATP) release, and αIIbß3 activation. ERp46 protein potentiated αIIbß3 activation, platelet aggregation, and ATP release, whereas inactive ERp46 inhibited these processes. ERp46 knockout mice had prolonged tail-bleeding times and decreased platelet accumulation in thrombosis models that was rescued by infusion of ERp46. ERp46-deficient platelets had decreased αIIbß3 activation, platelet aggregation, ATP release, and P-selectin expression. The defects were reversed by wild-type ERp46 and partially reversed by ERp46 containing any of the 3 active sites. Platelet aggregation stimulated by an αIIbß3-activating peptide was inhibited by the anti-ERp46 antibody and was decreased in ERp46-deficient platelets. ERp46 bound tightly to αIIbß3 by surface plasmon resonance but poorly to platelets lacking αIIbß3 and physically associated with αIIbß3 upon platelet activation. ERp46 mediated clot retraction and platelet spreading. ERp46 more strongly reduced disulfide bonds in the ß3 subunit than other PDIs and in contrast to PDI, generated thiols in ß3 independently of fibrinogen. ERp46 cleaved the Cys473-Cys503 disulfide bond in ß3, implicating a target for ERp46. Finally, ERp46-deficient platelets have decreased thiols in ß3, implying that ERp46 cleaves disulfide bonds in platelets. In conclusion, ERp46 is critical for platelet function and thrombosis and facilitates αIIbß3 activation by targeting disulfide bonds.
Asunto(s)
Hemostasis , Tiorredoxinas/metabolismo , Trombosis , Animales , Retículo Endoplásmico/metabolismo , Ratones , Ratones Noqueados , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Trombosis/genética , Trombosis/metabolismoRESUMEN
Lateral transmembrane (TM) helix-helix interactions between single-span membrane proteins play an important role in the assembly and signaling of many cell-surface receptors. Often, these helices contain two highly conserved yet distinct interaction motifs, arranged such that the motifs cannot be engaged simultaneously. However, there is sparse experimental evidence that dual-engagement mechanisms play a role in biological signaling. Here, we investigate the function of the two conserved interaction motifs in the TM domain of the integrin ß3-subunit. The first motif uses reciprocating "large-large-small" amino acid packing to mediate the interaction of the ß3 and αIIb TM domains and maintain the inactive resting conformation of the platelet integrin αIIbß3. The second motif, S-x3-A-x3-I, is a variant of the classical "G-x3-G" motif. Using site-directed mutagenesis, optical trap-based force spectroscopy, and molecular modeling, we show that S-x3-A-x3-I does not engage αIIb but rather mediates the interaction of the ß3 TM domain with the TM domain of the αv-subunit of the integrin αvß3. Like αIIbß3, αvß3 on circulating platelets is inactive, and in the absence of platelet stimulation is unable to interact with components of the subendothelial matrix. However, disrupting any residue in the ß3 S-x3-A-x3-I motif by site-directed mutations is sufficient to induce αvß3 binding to the αvß3 ligand osteopontin and to the monoclonal antibody WOW-1. Thus, the ß3-integrin TM domain is able to engage in two mutually exclusive interactions that produce alternate α-subunit pairing, creating two integrins with distinct biological functions.
Asunto(s)
Integrina alfaVbeta3/metabolismo , Proteínas de la Membrana/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Animales , Plaquetas/metabolismo , Células CHO , Membrana Celular/metabolismo , Secuencia Conservada , Cricetulus , Humanos , Integrina alfaVbeta3/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Dominios ProteicosRESUMEN
The fluorescent reporters commonly used to visualize proteins can perturb both protein structure and function. Recently, we found that 4-cyanotryptophan (4CN-Trp), a blue fluorescent amino acid, is suitable for one-photon imaging applications. Here, we demonstrate its utility in two-photon fluorescence microscopy by using it to image integrins on cell surfaces. Specifically, we used solid-phase peptide synthesis to generate CHAMP peptides labeled with 4-cyanoindole (4CNI) at their N-termini to image integrins on cell surfaces. CHAMP (computed helical anti-membrane protein) peptides spontaneously insert into membrane bilayers to target integrin transmembrane domains and cause integrin activation. We found that 4CNI labeling did not perturb the ability of CHAMP peptides to insert into membranes, bind to integrins, or cause integrin activation. We then used two-photon fluorescence microscopy to image 4CNI-containing integrins on the surface of platelets. Compared to a 4CNI-labeled scrambled peptide that uniformly decorated cell surfaces, 4CNI-labeled CHAMP peptides were present in discrete blue foci. To confirm that these foci represented CN peptide-containing integrins, we co-stained platelets with integrin-specific fluorescent monoclonal antibodies and found that CN peptide and antibody fluorescence coincided. Because 4CNI can readily be biosynthetically incorporated into proteins with little if any effect on protein structure and function, it provides a facile way to directly monitor protein behavior and protein-protein interactions in cellular environments. In addition, these results clearly demonstrate that the two-photon excitation cross section of 4CN-Trp is sufficiently large to make it a useful two-photon fluorescence reporter for biological applications.
Asunto(s)
Integrinas/metabolismo , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Triptófano/análogos & derivados , Aminoácidos/metabolismo , Plaquetas/metabolismo , Membrana Celular/metabolismo , Integrinas/fisiología , Péptidos/síntesis química , Péptidos/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Unión Proteica/fisiología , Dominios Proteicos/fisiología , Triptófano/síntesis química , Triptófano/químicaRESUMEN
Integrin αIIbß3, a transmembrane heterodimer, mediates platelet aggregation when it switches from an inactive to an active ligand-binding conformation following platelet stimulation. Central to regulating αIIbß3 activity is the interaction between the αIIb and ß3 extracellular stalks, which form a tight heterodimer in the inactive state and dissociate in the active state. Here, we demonstrate that alanine replacements of sensitive positions in the heterodimer stalk interface destabilize the inactive conformation sufficiently to cause constitutive αIIbß3 activation. To determine the structural basis for this effect, we performed a structural bioinformatics analysis and found that perturbing intersubunit contacts with favorable interaction geometry through substitutions to alanine quantitatively accounted for the degree of constitutive αIIbß3 activation. This mutational study directly assesses the relationship between favorable interaction geometry at mutation-sensitive positions and the functional activity of those mutants, giving rise to a simple model that highlights the importance of interaction geometry in contributing to the stability between protein-protein interactions.
Asunto(s)
Mutagénesis/fisiología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Alanina/química , Alanina/genética , Alanina/metabolismo , Regulación Alostérica/fisiología , Animales , Células CHO , Cricetinae , Cricetulus , Humanos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Unión Proteica/fisiología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismoRESUMEN
Binding of soluble fibrinogen to the activated conformation of the integrin αIIbß3 is required for platelet aggregation and is mediated exclusively by the C-terminal AGDV-containing dodecapeptide (γC-12) sequence of the fibrinogen γ chain. However, peptides containing the Arg-Gly-Asp (RGD) sequences located in two places in the fibrinogen Aα chain inhibit soluble fibrinogen binding to αIIbß3 and make substantial contributions to αIIbß3 binding when fibrinogen is immobilized and when it is converted to fibrin. Here, we employed optical trap-based nanomechanical measurements and computational molecular modeling to determine the kinetics, energetics, and structural details of cyclic RGDFK (cRGDFK) and γC-12 binding to αIIbß3. Docking analysis revealed that NMR-determined solution structures of cRGDFK and γC-12 bind to both the open and closed αIIbß3 conformers at the interface between the αIIb ß-propeller domain and the ß3 ßI domain. The nanomechanical measurements revealed that cRGDFK binds to αIIbß3 at least as tightly as γC-12. A subsequent analysis of molecular force profiles and the number of peptide-αIIbß3 binding contacts revealed that both peptides form stable bimolecular complexes with αIIbß3 that dissociate in the 60-120 pN range. The Gibbs free energy profiles of the αIIbß3-peptide complexes revealed that the overall stability of the αIIbß3-cRGDFK complex was comparable with that of the αIIbß3-γC-12 complex. Thus, these results provide a mechanistic explanation for previous observations that RGD- and AGDV-containing peptides are both potent inhibitors of the αIIbß3-fibrinogen interactions and are consistent with the observation that RGD motifs, in addition to AGDV, support interaction of αIIbß3 with immobilized fibrinogen and fibrin.
Asunto(s)
Fibrinógeno/química , Oligopéptidos/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Subunidades de Proteína/química , Sitios de Unión , Plaquetas/química , Plaquetas/metabolismo , Fibrinógeno/metabolismo , Humanos , Cinética , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Oligopéptidos/síntesis química , Oligopéptidos/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Unión Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Subunidades de Proteína/metabolismo , TermodinámicaRESUMEN
Fibrinogen binding to the integrin αIIbß3 mediates platelet aggregation and spreading on fibrinogen-coated surfaces. However,in vivoαIIbß3 activation and fibrinogen conversion to fibrin occur simultaneously, although the relative contributions of fibrinogenversusfibrin to αIIbß3-mediated platelet functions are unknown. Here, we compared the interaction of αIIbß3 with fibrin and fibrinogen to explore their differential effects. A microscopic bead coated with fibrinogen or monomeric fibrin produced by treating the immobilized fibrinogen with thrombin was captured by a laser beam and repeatedly brought into contact with surface-attached purified αIIbß3. When αIIbß3-ligand complexes were detected, the rupture forces were measured and displayed as force histograms. Monomeric fibrin displayed a higher probability of interacting with αIIbß3 and a greater binding strength. αIIbß3-fibrin interactions were also less sensitive to inhibition by abciximab and eptifibatide. Both fibrinogen- and fibrin-αIIbß3 interactions were partially inhibited by RGD peptides, suggesting the existence of common RGD-containing binding motifs. This assumption was supported using the fibrin variants αD97E or αD574E with mutated RGD motifs. Fibrin made from a fibrinogen γ'/γ' variant lacking the γC αIIbß3-binding motif was more reactive with αIIbß3 than the parent fibrinogen. These results demonstrate that fibrin is more reactive with αIIbß3 than fibrinogen. Fibrin is also less sensitive to αIIbß3 inhibitors, suggesting that fibrin and fibrinogen have distinct binding requirements. In particular, the maintenance of αIIbß3 binding activity in the absence of the γC-dodecapeptide and the α-chain RGD sequences suggests that the αIIbß3-binding sites in fibrin are not confined to its known γ-chain and RGD motifs.
Asunto(s)
Plaquetas/metabolismo , Fibrina/metabolismo , Fibrinógeno/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Plaquetas/citología , Humanos , Agregación Plaquetaria , Mapas de Interacción de ProteínasRESUMEN
αIIbß3 activation in platelets is followed by activation of the tyrosine kinase c-Src associated with the carboxyl terminus of the ß3 cytosolic tail. Exogenous peptides designed to interact with the αIIb transmembrane (TM) domain activate single αIIbß3 molecules in platelets by binding to the αIIb TM domain and causing separation of the αIIbß3 TM domain heterodimer. Here we asked whether directly activating single αIIbß3 molecules in platelets using the designed peptide anti-αIIb TM also initiates αIIbß3-mediated outside-in signaling by causing activation of ß3-associated c-Src. Anti-αIIb TM caused activation of ß3-associated c-Src and the kinase Syk, but not the kinase FAK, under conditions that precluded extracellular ligand binding to αIIbß3. c-Src and Syk are activated by trans-autophosphorylation, suggesting that activation of individual αIIbß3 molecules can initiate αIIbß3 clustering in the absence of ligand binding. Consistent with this possibility, incubating platelets with anti-αIIb TM resulted in the redistribution of αIIbß3 from a homogenous ring located at the periphery of discoid platelets into nodular densities consistent with clustered αIIbß3. Thus, these studies indicate that not only is resting αIIbß3 poised to undergo a conformational change that exposes its ligand-binding site, but it is poised to rapidly assemble into intracellular signal-generating complexes as well.
Asunto(s)
Plaquetas/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Quinasa Syk/metabolismo , Familia-src Quinasas/metabolismo , Proteína Tirosina Quinasa CSK , Humanos , Immunoblotting , Inmunoprecipitación , Fragmentos de Péptidos/farmacología , Fosforilación , Activación Plaquetaria , Agregación Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/químicaRESUMEN
It is currently believed that inactive tyrosine kinase c-Src in platelets binds to the cytoplasmic tail of the ß3 integrin subunit via its SH3 domain. Although a recent NMR study supports this contention, it is likely that such binding would be precluded in inactive c-Src because an auto-inhibitory linker physically occludes the ß3 tail binding site. Accordingly, we have re-examined c-Src binding to ß3 by immunoprecipitation as well as NMR spectroscopy. In unstimulated platelets, we detected little to no interaction between c-Src and ß3. Following platelet activation, however, c-Src was co-immunoprecipitated with ß3 in a time-dependent manner and underwent progressive activation as well. We then measured chemical shift perturbations in the (15)N-labeled SH3 domain induced by the C-terminal ß3 tail peptide NITYRGT and found that the peptide interacted with the SH3 domain RT-loop and surrounding residues. A control peptide whose last three residues where replaced with those of the ß1 cytoplasmic tail induced only small chemical shift perturbations on the opposite face of the SH3 domain. Next, to mimic inactive c-Src, we found that the canonical polyproline peptide RPLPPLP prevented binding of the ß3 peptide to the RT- loop. Under these conditions, the ß3 peptide induced chemical shift perturbations similar to the negative control. We conclude that the primary interaction of c-Src with the ß3 tail occurs in its activated state and at a site that overlaps with PPII binding site in its SH3 domain. Interactions of inactive c-Src with ß3 are weak and insensitive to ß3 tail mutations.
Asunto(s)
Plaquetas/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Transducción de Señal/fisiología , Familia-src Quinasas/metabolismo , Plaquetas/química , Plaquetas/citología , Proteína Tirosina Quinasa CSK , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Humanos , Péptidos/química , Péptidos/farmacología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Unión Proteica , Transducción de Señal/efectos de los fármacos , Dominios Homologos src , Familia-src Quinasas/química , Familia-src Quinasas/genéticaRESUMEN
In this issue of Blood, Kasirer-Friede and colleagues show that the adhesion and degranulation promoter protein (ADAP) promotes αIIbß3 activation by presenting the cytoplasmic proteins talin and kindlin to the ß3 cytoplasmic tail.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Citoesqueleto/metabolismo , Fibrinógeno/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Talina/metabolismo , Animales , HumanosRESUMEN
Blood platelets prevent bleeding after trauma by forming occlusive aggregates at sites of vascular injury. Platelet aggregation is mediated by the integrin heterodimer αIIbß3 and occurs when platelet agonists generated at the injury site convert αIIbß3 from its resting to its active conformation. Active αIIbß3 is then able to bind macromolecular ligands such as fibrinogen that crosslink adjacent platelets into hemostatic aggregates. Platelets circulate in a plasma milieu containing high concentrations of the principal αIIbß3 ligand fibrinogen. Thus, αIIbß3 activity is tightly regulated to prevent the spontaneous formation of platelet aggregates. αIIbß3 activity is regulated at least three levels. First, intramolecular interactions involving motifs located in the membrane-proximal stalk regions, transmembrane domains, and the membrane-proximal cytosolic tails of αIIb and ß3 maintain αIIbß3 in its inactive conformation. Transmembrane domain interactions appear particularly important because disrupting these interactions causes constitutive αIIbß3 activation. Second, the agonist-stimulated binding of the cytosolic proteins talin and kindlin-3 to the ß3 cytosolic tail rapidly causes αIIbß3 activation by disrupting the intramolecular interactions constraining αIIbß3 activity. Third, the strength of ligand binding to active αIIbß3 seems to be allosterically regulated. Thus, αIIbß3 exists in a minimum of three interconvertible states: an inactive (resting) state that does not interact with ligands and two active ligand binding states that differ in their affinity for fibrinogen and in the mechanical stability of fibrinogen complexes they form.
Asunto(s)
Plaquetas/metabolismo , Membrana Celular/metabolismo , Agregación Plaquetaria/fisiología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Secuencias de Aminoácidos , Animales , Humanos , Estructura Terciaria de ProteínaRESUMEN
Binding of the talin-1 FERM (4.1/ezrin/radixin/moesin) domain to the ß3 cytosolic tail causes activation of the integrin αIIbß3. The FERM domain also binds to acidic phospholipids. Although much is known about the interaction of talin-1 with integrins and lipids, the relative contribution of each interaction to integrin regulation and possible synergy between them remain to be clarified. Here, we examined the thermodynamic interplay between FERM domain binding to phospholipid bilayers and to its binding sites in the ß3 tail. We found that although both the F0F1 and F2F3 subdomains of the talin-1 FERM domain bind acidic bilayers, the full-length FERM domain binds with an affinity similar to F2F3, indicating that F0F1 contributes little to the overall interaction. When free in solution, the ß3 tail has weak affinity for the FERM domain. However, appending the tail to acidic phospholipids increased its affinity for the FERM domain by three orders of magnitude. Nonetheless, the affinity of the FERM for the appended tail was similar to its affinity for binding to bilayers alone. Thus, talin-1 binding to the ß3 tail is a ternary interaction dominated by a favorable surface interaction with phospholipid bilayers and set by lipid composition. Nonetheless, interactions between the FERM domain, the ß3 tail, and lipid bilayers are not optimized for a high-affinity synergistic interaction, even at the membrane surface. Instead, the interactions appear to be tuned in such a way that the equilibrium between inactive and active integrin conformations can be readily regulated.
Asunto(s)
Citosol/metabolismo , Integrina beta3/química , Integrina beta3/metabolismo , Membrana Dobles de Lípidos/metabolismo , Fosfolípidos/metabolismo , Talina/metabolismo , Proteínas del Citoesqueleto/química , Dextranos/metabolismo , Proteínas Inmovilizadas/metabolismo , Cinética , Proteínas de la Membrana/química , Proteínas de Microfilamentos/química , Modelos Moleculares , Fosfatidilinositol 4,5-Difosfato/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Resonancia por Plasmón de Superficie , Propiedades de SuperficieRESUMEN
Using a combined experimental and theoretical approach named binding-unbinding correlation spectroscopy (BUCS), we describe the two-dimensional kinetics of interactions between fibrinogen and the integrin αIIbß3, the ligand-receptor pair essential for platelet function during hemostasis and thrombosis. The methodology uses the optical trap to probe force-free association of individual surface-attached fibrinogen and αIIbß3 molecules and forced dissociation of an αIIbß3-fibrinogen complex. This novel approach combines force clamp measurements of bond lifetimes with the binding mode to quantify the dependence of the binding probability on the interaction time. We found that fibrinogen-reactive αIIbß3 pre-exists in at least two states that differ in their zero force on-rates (k(on1) = 1.4 × 10(-4) and k(on2) = 2.3 × 10(-4) µm(2)/s), off-rates (k(off1) = 2.42 and k(off2) = 0.60 s(-1)), and dissociation constants (K(d)(1) = 1.7 × 10(4) and K(d)(2) = 2.6 × 10(3) µm(-2)). The integrin activator Mn(2+) changed the on-rates and affinities (K(d)(1) = 5 × 10(4) and K(d)(2) = 0.3 × 10(3) µm(-2)) but did not affect the off-rates. The strength of αIIbß3-fibrinogen interactions was time-dependent due to a progressive increase in the fraction of the high affinity state of the αIIbß3-fibrinogen complex characterized by a faster on-rate. Upon Mn(2+)-induced integrin activation, the force-dependent off-rates decrease while the complex undergoes a conformational transition from a lower to higher affinity state. The results obtained provide quantitative estimates of the two-dimensional kinetic rates for the low and high affinity αIIbß3 and fibrinogen interactions at the single molecule level and offer direct evidence for the time- and force-dependent changes in αIIbß3 conformation and ligand binding activity, underlying the dynamics of fibrinogen-mediated platelet adhesion and aggregation.
Asunto(s)
Fibrinógeno/química , Modelos Químicos , Complejos Multiproteicos/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Plaquetas/química , Plaquetas/metabolismo , Fibrinógeno/metabolismo , Hemostasis/fisiología , Humanos , Cinética , Modelos Biológicos , Complejos Multiproteicos/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Trombosis/metabolismoRESUMEN
Interactions between transmembrane (TM) helices play an important role in the regulation of diverse biological functions. For example, the TM helices of integrins are believed to interact heteromerically in the resting state; disruption of this interaction results in integrin activation and cellular adhesion. However, it has been difficult to demonstrate the specificity and affinity of the interaction between integrin TM helices and to relate them to the activation process. To examine integrin TM helix associations, we developed a bacterial reporter system and used it to define the sequence motif required for helix-helix interactions in the beta (1) and beta (3) integrin subfamilies. The helices interact in a novel three-dimensional motif, the "reciprocating large-small motif" that is also observed in the crystal structures of unrelated proteins. Modest but specific stabilization of helix associations is realized via packing of complementary small and large groups on neighboring helices. Mutations destabilizing this motif activate native, full-length integrins. Thus, this highly conserved dissociable motif plays a vital and widespread role as an on-off switch that can integrate with other control elements during integrin activation.
Asunto(s)
Cadenas alfa de Integrinas/química , Cadenas beta de Integrinas/química , Secuencia de Aminoácidos , Línea Celular , Secuencia de Consenso , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Dimerización , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Humanos , Cadenas alfa de Integrinas/genética , Cadenas beta de Integrinas/genética , Células Jurkat , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Fragmentos de Péptidos/química , Regiones Promotoras Genéticas , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The integrin αIIbß3 is a transmembrane (TM) heterodimeric adhesion receptor that exists in equilibrium between resting and active ligand binding conformations. In resting αIIbß3, the TM and cytoplasmic domains of αIIb and ß3 form a heterodimer that constrains αIIbß3 in its resting conformation. To study the structure and dynamics of the cytoplasmic domain heterodimer, we prepared a disulfide-stabilized complex consisting of portions of the TM domains and the full cytoplasmic domains. NMR and hydrogen-deuterium exchange of this complex in micelles showed that the αIIb cytoplasmic domain is largely disordered, but it interacts with and influences the conformation of the ß3 cytoplasmic domain. The ß3 cytoplasmic domain consists of a stable proximal helix contiguous with the TM helix and two distal amphiphilic helices. To confirm the NMR structure in a membrane-like environment, we studied the ß3 cytoplasmic domain tethered to phospholipid bilayers. Hydrogen-deuterium exchange mass spectrometry, as well as circular dichroism spectroscopy, demonstrated that the ß3 cytoplasmic domain becomes more ordered and helical under these conditions, consistent with our NMR results. Further, these experiments suggest that the two distal helices associate with lipid bilayers but undergo fluctuations that would allow rapid binding of cytoplasmic proteins regulating integrin activation, such as talin and kindlin-3. Thus, these results provide a framework for understanding the kinetics and thermodynamics of protein interactions involving integrin cytoplasmic domains and suggest that such interactions act in a concerted fashion to influence integrin stalk separation and exposure of extracellular ligand binding sites.
Asunto(s)
Citoplasma/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Estructura Terciaria de Proteína , Animales , Sitios de Unión , Dicroismo Circular , Medición de Intercambio de Deuterio , Disulfuros/química , Disulfuros/metabolismo , Humanos , Cinética , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Unión Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Talina/química , Talina/metabolismo , TermodinámicaRESUMEN
Mesangial cells and podocytes express integrins α1ß1 and α2ß1, which are the two major collagen receptors that regulate multiple cellular functions, including extracellular matrix homeostasis. Integrin α1ß1 protects from glomerular injury by negatively regulating collagen production, but the role of integrin α2ß1 in renal injury is unclear. Here, we subjected wild-type and integrin α2-null mice to injury with adriamycin or partial renal ablation. In both of these models, integrin α2-null mice developed significantly less proteinuria and glomerulosclerosis. In addition, selective pharmacological inhibition of integrin α2ß1 significantly reduced adriamycin-induced proteinuria, glomerular injury, and collagen deposition in wild-type mice. This inhibitor significantly reduced collagen synthesis in wild-type, but not integrin α2-null, mesangial cells in vitro, demonstrating that its effects are integrin α2ß1-dependent. Taken together, these results indicate that integrin α2ß1 contributes to glomerular injury by positively regulating collagen synthesis and suggest that its inhibition may be a promising strategy to reduce glomerular injury and proteinuria.
Asunto(s)
Lesión Renal Aguda/patología , Doxorrubicina/farmacología , Integrina alfa2beta1/metabolismo , Glomérulos Renales/lesiones , Lesión Renal Aguda/metabolismo , Albuminuria/fisiopatología , Análisis de Varianza , Animales , Western Blotting , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Inmunohistoquímica , Integrina alfa2beta1/efectos de los fármacos , Pruebas de Función Renal , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Masculino , Células Mesangiales/efectos de los fármacos , Células Mesangiales/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Distribución Aleatoria , Receptores de Colágeno/metabolismoRESUMEN
Mortality from ischemic cardiac disease in adults has been dramatically reduced by the development of novel therapies for inhibiting platelet function. Circulating platelets are maintained in a resting state and are activated at sites of vascular injury by exquisitely controlled mechanisms, thereby maintaining vascular integrity without causing intravascular thrombosis. As it became clear that platelets play a central role in arterial thrombosis, the processes of platelet activation, adhesion, and aggregation became logical targets for the development of antithrombotic agents.
Asunto(s)
Inhibidores de Agregación Plaquetaria/uso terapéutico , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Abciximab , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Plaquetas/fisiología , Eptifibatida , Humanos , Fragmentos Fab de Inmunoglobulinas/farmacología , Fragmentos Fab de Inmunoglobulinas/uso terapéutico , Péptidos/farmacología , Péptidos/uso terapéutico , Inhibidores de Agregación Plaquetaria/farmacología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Tirofibán , Tirosina/análogos & derivados , Tirosina/farmacología , Tirosina/uso terapéuticoRESUMEN
There is a grave need for safer antiplatelet therapeutics to prevent heart attack and stroke. Agents targeting the interaction of platelets with the diseased vessel wall could impact vascular disease with minimal effects on normal hemostasis. We targeted integrin alpha(2)beta(1), a collagen receptor, because its overexpression is associated with pathological clot formation whereas its absence does not cause severe bleeding. Structure-activity studies led to highly potent and selective small-molecule inhibitors. Responses of integrin alpha(2)beta(1) mutants to these compounds are consistent with a computational model of their mode of inhibition and shed light on the activation mechanism of I-domain-containing integrins. A potent compound was proven efficacious in an animal model of arterial thrombosis, which demonstrates in vivo efficacy for inhibition of this platelet receptor. These results suggest that targeting integrin alpha(2)beta(1) could be a potentially safe, effective approach to long-term therapy for cardiovascular disease.