The N-terminal globular domain of the laminin α1 chain binds to α1β1 and α2β1 integrins and to the heparan sulfate-containing domains of perlecan

The N-terminal domains VI plus V (62 kDa) and V alone (43 kDa) of the laminin α1 chain were obtained as recombinant products and shown to be folded into a native form by electron microscopy and immunological assays. Domain VI alone, which corresponds to an LN module, did not represent an autonomously folding unit in mammalian cells, however. Fragment α1VI/V, but not fragment α1V, bound to purified α1β1 and α2β1 integrins, to heparin, and to heparan sulfate-substituted domains I and V of perlecan. This localized the binding activities to the LN module, which contains two basic sequences suitable for heparin interactions.     

The αβ complexes of ATP synthase: the α3β3 oligomer and α1β1 protomer

The basic structures of the catalytic portion (F₁, ₃₃) of ATP synthase are the ₃₃ hexamer (oligomer with cooperativity) and ₁₁ heterodimer (protomer). These were reconstituted from the and subunits of thermophilic F₁ (TF₁), and the ₃₃ hexamer was crystallized. On electrophoresis, both the dimer and hexamer showed bands with ATPase activity. Using the dimer and hexamer, we studied the nucleotide-dependent rapid molecular dynamics. The formation of the hexamer required neither nucleotide nor Mg. The hexamer was dissociated into the dimer in the presence of MgADP, while the dimer was associated into the hexamer in the presence of MgATP. The hexamer, like mitochondrial F₁ and TF₁, showed two kinds of ATPase activity: one was cooperative and was inhibited by only one BzADP per hexamer, and the other was inhibited by three BzADP per hexamer.     

Structural Basis of the Differential Binding of Engineered Knottins to Integrins αVβ3 and α5β1

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Synthesis and initial evaluation of novel,non-peptidic antagonists of the αv-integrins αvβ3 and αvβ5

The discovery, synthesis and preliminary SAR of a novel class of non-peptidic antagonists of the α_v-integrins α_vβ₃ and α_vβ₅ is described. High-throughput screening of an extensive series of ECLiPS? compound libraries led to the identification of compound 1 as a dual inhibitor of the α_v-integrins α_vβ₃ and α_vβ₅. Optimization of compound 1 involving, in part, introduction of two novel constraints led to the discovery of compounds 15a and 15b with reduced PSA and much improved potency for both the α_vβ₃ and α_vβ₅ integrins. Compounds 15a and 15b were shown to have promising activity in functional cellular assays and compound 15a also exhibited a promising Caco-2 permeability profile.     

Activation of the integrins α5β1 and αvβ3 and focal adhesion kinase (FAK) during arteriogenesis

Migration and proliferation of smooth muscle cells (SMC) are important events during arteriogenesis, but the underlying mechanism is still only partially understood. The present study investigates the expression of integrins alpha 5 beta 1 and v beta 3 as well as focal adhesion kinase (FAK) and phosphorylated FAK (pY397), key mediators for cell migration and proliferation, in collateral vessels (CV) in rabbit hind limbs induced by femoral ligation or an arteriovenous (AV) shunt created between the distal femoral artery stump and the accompanying femoral vein by confocal immunofluorescence. In addition, the effect of the extracellular matrix components fibronectin (FN), laminin (LN), and Matrigel on expression of these focal adhesion molecules proliferation was studied in cultured SMCs. We found that: (1) in normal vessels (NV), both integrins alpha 5 beta 1 and alpha v beta 3 were mainly expressed in endothelial cells, very weak in smooth muscle cells (SMC); (2) in CVs, both alpha 5 beta 1 and alpha v beta 3 were significantly upregulated (P < 0.05); this was more evident in the shunt-side CVs, 1.5 and 1.3 times higher than that in the ligation side, respectively; (3) FAK and FAK(py397) were expressed in NVs and CVs in a similar profile as was alpha 5 beta 1 and alpha v beta 3; (4) in vitro SMCs cultured on fibronectin (overexpressed in collaterals) expressed higher levels of FAK, FAK (pY397), alpha 5 beta 1, and alpha v beta 3 than on laminin, whereas SMCs growing inside Matrigel expressed little of these proteins and showed no proliferation. In conclusion, our data demonstrate for the first time that the integrin-FAK signaling axis is activated in collateral vessels and that altered expression of FN and LN may play a crucial role in mediating the integrin-FAK signaling pathway activation. These findings explain a large part of the positive remodeling that collateral vessels undergo under the influence of high fluid shear stress.     

Physiological and Pathological Roles of α3β1 Integrin

α3β1 integrin has been considered to be a mysterious adhesion molecule due to the pleiotropy in its ligand-binding specificity. However, recent studies have identified laminin isoforms as high-affinity ligands for this integrin, and demonstrated that α3β1 integrin plays a number of essential roles in development and differentiation, mainly by mediating the establishment and maintenance of epithelial tissues. Furthermore, α3β1 integrin is also implicated in many other biological phenomena, including cell growth and apoptosis, angiogenesis and neural functions. This integrin receptor forms complexes with various other membrane proteins, such as the transmembrane-4 superfamily proteins (tetraspanins), cytoskeletal proteins and signaling molecules. Recently, lines of evidence have been reported showing that complex formation regulates integrin functions in cell adhesion and migration, signal transduction across cell membranes, and cytoskeletal organization. In addition to these roles in physiological processes, α3β1 integrin performs crucial functions in various pathological processes, especially in wound healing, tumor invasion and metastasis, and infection by pathogenic microorganisms.This revised version was published online in June 2005 with a corrected cover date.     

Structural and functional insights into the heme-binding domain of the human soluble guanylate cyclase α2 subunit and heterodimeric α2β1

Soluble guanylate cyclase (sGC) mediates NO signaling for a wide range of physiological effects in the cardiovascular system and the central nervous system. The α1β1 isoform is ubiquitously distributed in cytosolic fractions of tissues, whereas α2β1 is mainly found in the brain. The major occurrence and the unique characteristic of human sGC α2β1 indicate a special role in the mediation of neuronal communication. We have efficiently purified and characterized the recombinant heme-binding domain of the human sGC α2 subunit (hsGC α2(H)) and heterodimeric α2β1 (hsGC β1(H)-α2(H)) by UV-vis spectroscopy, circular dichrosim spectroscopy, EPR spectroscopy, and homology modeling. The heme dissociation and related NO/CO binding/dissociation of both hsGC α2(H) and hsGC β1(H)-α2(H) were investigated. The two truncated proteins interact with heme noncovalently. The CO binding affinity of hsGC α2(H) is threefold greater than that of human sGC α1(H), whereas the dissociation constant k (1) for dissociation of NO from hsGC α2(H) is sevenfold larger than that for dissociation of NO from hsGC α1(H), although k (2) is almost identical. The results indicate that in comparison with the α1β1 isoform, the brain α2β1 isoform exhibits a distinctly different CO/NO affinity and binding rate in favor of NO signaling, and this is consistent with its physiological role in the activation and desensitization. Molecular modeling and sequence alignments are consistent with the hypothesis that His105 contributes to the different CO/NO binding properties of different isoforms. This valuable information is helpful to understand the molecular mechanism by which human sGC α2β1 mediates NO/CO signaling.     

Affinity of different α1-agonists and antagonists for the α1-adrenoceptors of rabbit and rat liver membranes

In membranes prepared from rabbit liver, competition with [³H] prazosin by different α₁-agonists and antagonists revealed different affinities in comparison to the results obtained on rat liver membranes, and showed a good correlation with the affinity of the same compounds for the cloned α_1c-adrenoceptor subtype. The potencies observed on rat liver membranes were well correlated with the affinity observed for the cloned α_1b-adrenoceptors. These results confirm that rabbit and rat liver membranes preparations can be utilized to evaluate the affinity of compounds for these α₁-adrenergic subtypes.     

Interaction of αVβ3 and αVβ6 Integrins with Human Parechovirus 1

Human parechovirus (HPEV) infections are very common in early childhood and can be severe in neonates. It has been shown that integrins are important for cellular infectivity of HPEV1 through experiments using peptide blocking assays and function-blocking antibodies to α_V integrins. The interaction of HPEV1 with α_V integrins is presumably mediated by a C-terminal RGD motif in the capsid protein VP1. We characterized the binding of integrins α_Vβ₃ and α_Vβ₆ to HPEV1 by biochemical and structural studies. We showed that although HPEV1 bound efficiently to immobilized integrins, α_Vβ₆ bound more efficiently than α_Vβ₃ to immobilized HPEV1. Moreover, soluble α_Vβ₆, but not α_Vβ₃, blocked HPEV1 cellular infectivity, indicating that it is a high-affinity receptor for HPEV1. We also showed that HPEV1 binding to integrins in vitro could be partially blocked by RGD peptides. Using electron cryo-microscopy and image reconstruction, we showed that HPEV1 has the typical T=1 (pseudo T=3) organization of a picornavirus. Complexes of HPEV1 and integrins indicated that both integrin footprints reside between the 5-fold and 3-fold symmetry axes. This result does not match the RGD position predicted from the coxsackievirus A9 X-ray structure but is consistent with the predicted location of this motif in the shorter C terminus found in HPEV1. This first structural characterization of a parechovirus indicates that the differences in receptor binding are due to the amino acid differences in the integrins rather than to significantly different viral footprints.Picornaviruses consist of a positive-sense, single-stranded infectious RNA genome of approximately 7.3 kb enclosed in a capsid composed of 60 copies of each of the three or four capsid proteins (VP1 to VP4). Human parechovirus 1 (HPEV1) is a member of the Parechovirus genus of the Picornaviridae family (38, 70). There are currently eight completely sequenced human parechovirus types and 14 described types (4, 19, 24, 30, 38, 39, 51, 58, 78). In addition, the Parechovirus genus currently has four Ljungan virus members that infect rodents. HPEV1 exhibits several distinct molecular characteristics compared to other picornaviruses (38, 71). These include the lack of the maturation cleavage of the capsid proteins VP0 to VP4 (N-terminal) and VP2 (C-terminal), existence of an approximately 30-amino-acid-long extension to the N terminus of VP3, a unique nonstructural protein 2A, and a 5′ untranslated region that is more closely related to picornaviruses infecting animals than those infecting humans.HPEV infections are common during the first years of life and are often mild or asymptomatic (20, 28, 42, 73, 80). Recently, a number of new types have been identified, and their prevalence in stool samples, for example, highlights their clinical importance. Normally, they cause gastroenteritis and respiratory infections, but severe illnesses, such as infections of the central nervous system, generalized infections of neonates, and myocarditis, have also been associated with HPEV infections (1, 8, 10, 28, 80). Currently, the role of the unique molecular, structural, and antigenic characteristics of HPEVs in the pathogenesis of infection is unknown.HPEV types 1, 2, 4, 5, and 6 are known to possess an RGD motif near the C terminus of VP1 that is known to facilitate binding of cellular ligands (e.g., fibronectin) to α_v integrins. The motif is in an analogous position to motifs in coxsackievirus A9 (CAV9) and echovirus 9 (EV9; Barty strain) (Fig. ​(Fig.1).1). The role of the RGD sequence in cellular entry and subsequent replication of HPEV1 has been shown through blocking assays with RGD-containing peptides, mutation of the sequence, and function-blocking antibodies to α_v integrins (11, 43, 62, 71). These results strongly suggested that α_v integrins play a central role in the initiation of HPEV1 infection. Direct involvement of α_v integrins in the infectious entry of HPEV1 was further confirmed by overexpression of human α_vβ₁ and α_vβ₃ integrins in Chinese hamster ovary (CHO) cells, allowing successful virus infection (74). There are no reports yet on the identification of receptors for the HPEV types lacking the RGD motif (HPEV3, HPEV7, and HPEV8) (19, 39, 51).Open in a separate windowFIG. 1.Sequence alignments. Amino acid sequence alignment of the viral coat protein VP1 from different picornaviruses with the CAV9 secondary structure derived from the atomic model displayed above the alignment (34). The columns boxed in blue with red letters signify similarity, and the red column signifies identity. There is limited similarity between HPEV and other picornaviruses. C-terminal RGD motifs are boxed in red.Although the crystal structures of several picornaviruses have been determined (3, 26, 34, 35, 44, 57, 59, 65, 68, 72) and the receptor interactions have been studied in detail by X-ray crystallography, electron cryo-microscopy (cryo-EM), and three-dimensional (3D) image reconstruction (6, 9, 23, 31, 32, 47, 83), there is no structural information available for the parechoviruses or parechovirus-receptor complexes. Here, we compare the binding of α_Vβ₃ and α_Vβ₆ to HPEV1 in vitro by biochemical assays and determine the structures of HPEV1 and the corresponding HPEV1-integrin complexes.     

Hydroxylysine in the N-terminal regions of the α1- and α2-chains of various collagens

The degree of hydroxylation of the lysine residue located in both alpha(1)- and alpha(2)-chains of collagen in the N-terminal, non-helical telopeptide region of the molecule has been determined in collagen from various sources after isolation of the peptides (alpha(1)- and alpha(2)-CB1) that contain the lysine residue in question and are obtained by cyanogen bromide cleavage of collagen alpha(1)- and alpha(2)-chains respectively. As with collagen from chick tibia, bone collagens from rat tibia and femur and embryonic chick frontal bone, have a high degree of hydroxylation (approx. 50% or more) of the lysine residue in both alpha(1)- and alpha(2)-CB1 peptides. This is in contrast with the lack of hydroxylation of this residue in both alpha(1)- and alpha(2)-chains of all skin collagens so far examined. The presence of hydroxylysine in alpha(1)- and alpha(2)-CB1 peptides from tendon collagen is also indicated. In rat tail tendon collagen the amount of hydroxylation is only slight but in the much less soluble tendon collagen from embryonic chick leg tendons, approximately one-third of the lysine is hydroxylated.     

Mechanism of the cyanide-catalyzed oxidation of α-ketoaldehydes and α-ketoalcohols

Cyanide catalyzes the reduction of dioxygen or of ferricytochrome c by dihydroxyacetone phosphate. The rapid initial phase of these reactions, but not the subsequent slow phase, was augmented by incubating the triose phosphate aerobically or anaerobically at pH 9.0 prior to adding the cyanide. The aerobic incubation, which was most effective, was associated with a decline in enediol, whereas the less effective anaerobic incubation was accompanied by an increase in enediol content. This suggested that the α-ketoaldehyde product of autoxidation of the enediol, rather than the enediol itself, was responsible for the rapid phase reaction which followed addition of cyanide. This was confirmed by exploring the cyanide-catalyzed oxidation of the α-ketoaldehyde, phenylglyoxal. The inhibitory effect of the manganese-containing superoxide dismutase indicated that O₂⁻ was a kinetically important intermediate of the rapid phase reaction. A reaction mechanism is proposed which is consistent with the results presented.     

Cell adhesion to anosmin via α5β1, α4β1, and α9β1 integrins

Anosmin is an extracellular matrix protein, and genetic defects in anosmin result in human Kallmann syndrome. It functions in neural crest formation, cell adhesion, and neuronal migration. Anosmin consists of multiple domains, and it has been reported to bind heparan sulfate, FGF receptor, and UPA. In this study, we establish cell adhesion/spreading assays for anosmin and use them for antibody inhibition analyses to search for an integrin adhesion receptor. We find that α5β1, α4β1, and α9β1 integrins are needed for effective adhesive receptor function in cell adhesion and cell spreading on anosmin; adhesion is inhibited by both RGD and α4β1 CS1-based peptides. This identification of anosmin-integrin adhesion receptors should facilitate studies of anosmin function in cell and developmental biology.     

The TF1-ATPase and ATPase activities of assembled α3β3γ, α3β3γδ, and α3β3γε complexes are stimulated by low and inhibited by high concentrations of rhodamine 6G whereas the dye only inhibits the α3β3, and α3β3δ complexes

The ATPase activity of the F₁-ATPase from the thermophilic bacterium PS3 is stimulated at concentrations of rhodamine 6G up to about 10 µM where 70% stimulation is observed at 36°C. Half maximal stimulation is observed at about 3 µM dye. At rhodamine 6G concentrations greater than 10 µM, ATPase activity declines with 50% inhibition observed at about 75 µM dye. The ATPase activities of the ₃₃ and ₃₃ complexes assembled from isolated subunits of TF₁ expressed inE. coli deleted of theunc operon respond to increasing concentrations of rhodamine 6G nearly identically to the response of TF₁. In contrast, the ATPase activities of the ₃₃ and ₃₃ complexes are only inhibited by rhodamine 6G with 50% inhibition observed, respectively, at 35 and 75 µM dye at 36°C. The ATPase activity of TF₁ is stimulated up to 4-fold by the neutral detergent, LDAO. In the presence of stimulating concentrations of LDAO, the ATPase activity of TF₁ is no longer stimulated by rhodamine 6G, but rather, it is inhibited with 50% inhibition observed at about 30 µM dye at 30°C. One interpretation of these results is that binding of rhodamine 6G to a high-affinity site on TF₁ stimulates ATPase activity and unmasks a low-affinity, inhibitory site for the dye which is also exposed by LDAO.     

Expression of the c-Met Proto-Oncogene and Integrin α5β1 in Human Gastric Cardia Adenocarcinoma

The hepatocyte growth factor receptor c-Met, a receptor tyrosine kinase, and Integrin α5β1, one of the main ECM receptors of hepatocytes have been reported to play important roles in tumor growth by activating mitogenic signaling pathways. In human gastric cardia adenocarcinoma, however, expression of the c-Met and Integrin α5β1 have not been reported. Here we examined the mRNA levels and protein expressions of these two genes and their relationship in human normal gastric cardia mucosa and primary carcinomas. Quantitative real-time PCR was used to analyze the mRNA expression of both c-Met and Integrin α5β1. The relationship between c-Met and Integrin α5β1 expression and the histologic characteristics of tumors were studied. Western blot analysis was performed to investigate the presence of c-Met and Integrin α5β1. The expression patterns of c-Met and Integrin α5β1 in 45 frozen slides of cardia adenocarcinoma were identified by immunohistochemistry. Our results indicate that the expression of c-Met and of Integrin α5β1 was significantly associated with tumor differentiation, TNM, and metastasis via the lymphogenic route. A significant positive correlation was also found between c-Met and Integrin α5β1 mRNA expression, suggesting that expression of c-Met and Integrin α5β1 has mechanical significance in the early stages of human gastric cardia adenocarcinoma.     

Structure and Binding Interface of the Cytosolic Tails of αXβ2 Integrin

Background

Integrins are signal transducer proteins involved in a number of vital physiological processes including cell adhesion, proliferation and migration. Integrin molecules are hetero-dimers composed of two distinct subunits, α and β. In humans, 18 α and 8 β subunits are combined into 24 different integrin molecules. Each of the subunit comprises a large extracellular domain, a single pass transmembrane segment and a cytosolic tail (CT). The CTs of integrins are vital for bidirectional signal transduction and in maintaining the resting state of the receptors. A large number of intracellular proteins have been found to interact with the CTs of integrins linking integrins to the cytoskeleton.

Methodology/Principal Findings

In this work, we have investigated structure and interactions of CTs of the leukocyte specific integrin αXβ2. We determined the atomic resolution structure of a myristoylated CT of αX in perdeuterated dodecylphosphocholine (DPC) by NMR spectroscopy. Our results reveal that the 35-residue long CT of αX adopts an α-helical conformation for residues F4-N17 at the N-terminal region. The remaining residues located at the C-terminal segment of αX delineate a long loop of irregular conformations. A segment of the loop maintains packing interactions with the helical structure by an extended non-polar surface of the αX CT. Interactions between αX and β2 CTs are demonstrated by ¹⁵N-¹H HSQC NMR experiments. We find that residues constituting the polar face of the helical conformation of αX are involved in interactions with the N-terminal residues of β2 CT. A docked structure of the CT complex indicates that a network of polar and/or salt-bridge interactions may sustain the heteromeric interactions.

Conclusions/Significance

The current study provides important insights into the conservation of interactions and structures among different CTs of integrins.     

Short form of α9 promotes α9β1 integrin-dependent cell adhesion by modulating the function of the full-length α9 subunit

The α9β1 integrin is a multifunctional receptor that interacts with a variety of ligands including vascular cell adhesion molecule 1, tenascin-C, and osteopontin. A 2.3-kb truncated form of α9 integrin subunit cDNA was identified by searching the Medline database. This splice variant, which we called the short form of α9 integrin (SFα9), encodes a 632-aa isoform lacking transmembrane and cytoplasmic domains, and its authentic expression was verified by PCR and Western blotting. SFα9 is expressed on the cell surface but cannot bind ligand in the absence of the full-length α9 subunit. Over-expression of SFα9 in cells expressing full-length α9 promotes α9-dependent cell adhesion. This promoting effect of SFα9 requires the authentic cytoplasmic domain of the co-expressed full-length α9 subunit. Thus, SFα9 is a novel functional modulator of α9β1 integrin by inside-out signaling.     

Modeling and Molecular Dynamics of HPA-1a and -1b Polymorphisms: Effects on the Structure of the β3 Subunit of the αIIbβ3 Integrin

Background

The HPA-1 alloimmune system carried by the platelet integrin αIIbβ3 is the primary cause of alloimmune thrombocytopenia in Caucasians and the HPA-1b allele might be a risk factor for thrombosis. HPA-1a and -1b alleles are defined by a leucine and a proline, respectively, at position 33 in the β3 subunit. Although the structure of αIIbβ3 is available, little is known about structural effects of the L33P substitution and its consequences on immune response and integrin functions.

Methodology/Principal Findings

A complete 3D model of the L33-β3 extracellular domain was built and a P33 model was obtained by in silico mutagenesis. We then performed molecular dynamics simulations. Analyses focused on the PSI, I-EGF-1, and I-EGF-2 domains and confirmed higher exposure of residue 33 in the L33 β3 form. These analyses also showed major structural flexibility of all three domains in both forms, but increased flexibility in the P33 β3 form. The L33P substitution does not alter the local structure (residues 33 to 35) of the PSI domain, but modifies the structural equilibrium of the three domains.

Conclusions

These results provide a better understanding of HPA-1 epitopes complexity and alloimmunization prevalence of HPA-1a. P33 gain of structure flexibility in the β3 knee may explain the increased adhesion capacity of HPA-1b platelets and the associated thrombotic risk. Our study provides important new insights into the relationship between HPA-1 variants and β3 structure that suggest possible effects on the alloimmune response and platelet function.     

Cyclic RGD peptides interfere with binding of the Helicobacter pylori protein CagL to integrins αVβ3 and α5β1

The human pathogen Helicobacter pylori that may cause different gastric diseases exploits integrins for infection of gastric cells. The H. pylori protein CagL present on the outer region of the type IV secretion pilus contains an RGD sequence (-Arg-Gly-Asp-) that enables binding to cells presenting integrins α5β1 and αVβ3. This interaction can be inhibited with conformationally designed cyclic RGD peptides derived from the CagL epitope -Ala-Leu-Arg-Gly-Asp-Leu-Ala-. The inhibition of the CagL-αVβ3 interaction by different RGD peptides strongly suggests the importance of the RGD motif for CagL binding. CagL point mutants (RAD, RGA) show decreased affinity to integrin αVβ3. Furthermore, structure-activity relationship studies with cyclic RGD peptides in a spatial screening approach show the distinct influence of the three-dimensional arrangement of RGD motif on the ability to interfere with this interaction. Resulting from these studies, similar structural requirements for the CagL epitope as previously suggested for other ligands of integrin αVβ3 are proposed.     

Selective binding and lateral clustering of α5β1 and αvβ3 integrins: Unraveling the spatial requirements for cell spreading and focal adhesion assembly

ABSTRACT

Coordination of the specific functions of α5β1 and αvβ3 integrins is crucial for the precise regulation of cell adhesion, spreading and migration, yet the contribution of differential integrin-specific crosstalk to these processes remains unclear. To determine the specific functions of αvβ3 and α5β1 integrins, we used nanoarrays of gold particles presenting immobilized, integrin-selective peptidomimetic ligands. Integrin binding to the peptidomimetics is highly selective, and cells can spread on both ligands. However, spreading is faster and the projected cell area is greater on α5β1 ligand; both depend on ligand spacing. Quantitative analysis of adhesion plaques shows that focal adhesion size is increased in cells adhering to αvβ3 ligand at 30 and 60 nm spacings. Analysis of αvβ3 and α5β1 integrin clusters indicates that fibrillar adhesions are more prominent in cells adhering to α5β1 ligand, while clusters are mostly localized at the cell margins in cells adhering to αvβ3 ligand. αvβ3 integrin clusters are more pronounced on αvβ3 ligand, though they can also be detected in cells adhering to α5β1 ligand. Furthermore, α5β1 integrin clusters are present in cells adhering to α5β1 ligand, and often colocalize with αvβ3 clusters. Taken together, these findings indicate that the activation of αvβ3 integrin by ligand binding is dispensable for initial adhesion and spreading, but essential to formation of stable focal adhesions.     

Cholesterol Dependence of Collagen and Echovirus 1 Trafficking along the Novel α2β1 Integrin Internalization Pathway

We have previously shown that soluble collagen and a human pathogen, echovirus 1 (EV1) cluster α2β1 integrin on the plasma membrane and cause their internalization into cytoplasmic endosomes. Here we show that cholesterol plays a major role not only in the uptake of α2β1 integrin and its ligands but also in the formation of α2 integrin-specific multivesicular bodies (α2-MVBs) and virus infection. EV1 infection and α2β1 integrin internalization were totally halted by low amounts of the cholesterol-aggregating drugs filipin or nystatin. Inhibition of cholesterol synthesis and accumulation of lanosterol after ketoconazole treatment inhibited uptake of collagen, virus and clustered integrin, and prevented formation of multivesicular bodies and virus infection. Loading of lipid starved cells with cholesterol increased infection to some extent but could not completely restore EV1 infection to control levels. Cold Triton X-100 treatment did not solubilize the α2-MVBs suggesting, together with cholesterol labeling, that the cytoplasmic endosomes were enriched in detergent-resistant lipids in contrast to αV integrin labeled control endosomes in the clathrin pathway. Cholesterol aggregation leading to increased ion permeability caused a significant reduction in EV1 uncoating in endosomes as judged by sucrose gradient centrifugation and by neutral red-based uncoating assay. In contrast, the replication step was not dependent on cholesterol in contrast to the reports on several other viruses. In conclusion, our results showed that the integrin internalization pathway is dependent on cholesterol for uptake of collagen, EV1 and integrin, for maturation of endosomal structures and for promoting EV1 uncoating. The results thus provide novel information for developing anti-viral strategies and more insight into collagen and integrin trafficking.