Low-affinity State Research Articles

Regulatory Role of the Extracellular α-Tail/β-Tail Interaction in αIIbβ3 Integrin Activation

AbstractAbstract 3199The platelet αIIbβ3 integrin changes its conformation to increase the affinity for ligands upon platelet activation. It has been shown that binding of the intracellular proteins to the cytoplasmic tails activate αIIbβ3 by disrupting the endogenous interaction between the two cytoplasmic tails in inside-out signaling. However, the mechanism how separation of the cytoplasmic tails initiate structural rearrangement of the extracellular domains has not been investigated. In αIIbβ3 crystal structure, the α-extracellular tail consisting of thigh, calf-1, and calf-2 domains makes large interface with the β-extracellular tail consisting of EGF-2, -3, -4, and βT domains. We have previously shown that clasp formation between the two extracellular tails completely abrogated αIIbβ3 activation induced by the cytoplasmic tail truncation. This suggests that separation of the two extracellular tails is essential for activation induced by inside-out signaling. To examine whether this is the case, N-glycan binding sites consisting of the N-X-T/S motif were introduced in αIIb amino acid residues located in the thigh/EGF-2, calf-1/EGF-3, or calf-2/βT interfaces. Attachment of a bulky N-glycan to these sites would prevent endogenous interface formation, thus forces the tails to separate from each other. When expressed in CHO cells, Q513NKT mutation in the thigh, M660NRT mutation in the calf-1, V760NVT mutation in the calf-2 domain all induced robust fibrinogen binding without any activators. Attachment of N-glycan was confirmed by SDS-PAGE analysis on the αIIb fragments carrying these mutations. This activation was inhibited by stabilizing the β-head/β-tail interface or by limiting the αIIb movement with artificially introduced disulfides, both of which prevents integrin from assuming the extended conformer. Prevention of the hybrid domain swing-out also completely inhibited activation. These results suggest that separation of the tails activate αIIbβ3 by inducing integrin extension. To confirm if endogenous α-tail/β-tail interaction indeed keeps integrin in low affinity state, αIIb amino acid residues that participate in the interface formation were mutated and the effect on fibrinogen binding was examined. Among the mutants, the K514A in the thigh, R671A in the calf-1, and R751A in the calf-2 domains induced only weak activation. However, combining these mutations resulted in robust activation. This activation was completely inhibited by the C-terminal clasp formation. Taken together, these results suggest that the α-tail/β-tail interface is kept by a group of key interdomain interaction that individually is not strong enough to do so. In conclusion, the α-tail/β-tail interface interaction regulates integrin activation by keeping integrin in low affinity state. Disruption of the interaction induces separation of the extracellular tails and activates integrin by initiating the structural rearrangement from the low affinity bent conformer to the high affinity extended conformer. Disclosures:No relevant conflicts of interest to declare.

Current State of Agonist Radioligands for Imaging of Brain Dopamine D2/D3 Receptors In Vivo with Positron Emission Tomography

Dopamine (DA) is known to play an important role in numerous brain functions and has been suggested to be involved in several neuropsychiatric and neurological disorders. From early on, positron emission tomography (PET) studies of the DA system have yielded high interest. Currently, several aspects of the functionality of DA neurons can be imaged, including the DA synthesis rate and the expression of DA-related proteins (receptors and transporters). A more recent application of radioligands targeting DA receptors is to study the endogenous neurotransmitter levels in vivo in brain. In vitro binding studies have suggested that in general DA receptors, as many other G-protein coupled receptors, exist in two affinity states for agonist binding. The high affinity state is thought to represent the functional state of the receptor, and the proportion between high and low affinity states may change with the development of disease. PET imaging with agonist radioligands may provide information on the existence of the high affinity state in vivo. In addition, DA receptor agonist radioligands may be superior tools for measuring changes in endogenous DA levels as antagonist radioligands inherently bind to both the low and the high affinity state. This review primarily summarizes the present status of the D(2)/D(3) receptor agonist PET radioligands. In addition several PET studies evaluating the utility of the agonist concept are discussed.

Modulation of Integrin Activation by an Entropic Spring in the β-Knee

We show that the length of a loop in the β-knee, between the first and second cysteines (C1-C2) in integrin EGF-like (I-EGF) domain 2, modulates integrin activation. Three independent sets of mutants, including swaps among different integrin β-subunits, show that C1-C2 loop lengths of 12 and longer favor the low affinity state and masking of ligand-induced binding site (LIBS) epitopes. Shortening length from 12 to 4 residues progressively increases ligand binding and LIBS epitope exposure. Compared with length, the loop sequence had a smaller effect, which was ascribable to stabilizing loop conformation, and not interactions with the α-subunit. The data together with structural calculations support the concept that the C1-C2 loop is an entropic spring and an emerging theme that disordered regions can regulate allostery. Diversity in the length of this loop may have evolved among integrin β-subunits to adjust the equilibrium between the bent and extended conformations at different set points.

2E8 Binds to the High Affinity I-domain in a Metal Ion-dependent Manner: A SECOND GENERATION MONOCLONAL ANTIBODY SELECTIVELY TARGETING ACTIVATED LFA-1

The activation of leukocyte function-associated antigen-1 (LFA-1) plays a critical role in regulating immune responses. The metal ion-dependent adhesion site on the I-domain of LFA-1 α(L) subunit is the key recognition site for ligand binding. Upon activation, conformation changes in the I-domain can lead LFA-1 from the low affinity state to the high affinity (HA) state. Using the purified HA I-domain locked by disulfide bonds for immunization, we developed an mAb, 2E8, that specifically binds to cells expressing the HA LFA-1. The surface plasmon resonance analysis has shown that 2E8 only binds to the HA I-domain and that the dissociation constant (K(D)) for HA I-domain is 197 nm. The binding of 2E8 to the HA I-domain is metal ion-dependent, and the affinity decreased as Mn(2+) was replaced sequentially by Mg(2+) and Ca(2+). Surface plasmon resonance analysis demonstrates that 2E8 inhibits the interaction of HA I-domain and ICAM-1. Furthermore, we found that 2E8 can detect activated LFA-1 on both JY and Jurkat cells using flow cytometry and parallel plate adhesion assay. In addition, 2E8 inhibits JY cell adhesion to human umbilical vein endothelial cells and homotypic aggregation. 2E8 treatment reduces the proliferation of both human CD4(+) and CD8(+) T cells upon OKT3 stimulation without the impairment of their cytolytic function. Taken together, these data demonstrate that 2E8 is specific for the high affinity form of LFA-1 and that 2E8 inhibits LFA-1/ICAM-1 interactions. As a novel activation-specific monoclonal antibody, 2E8 is a potentially useful reagent for blocking high affinity LFA-1 and modulating T cell activation in research and therapeutics.

Adenosine A2A Receptor Signaling and Golf Assembly Show a Specific Requirement for the γ7 Subtype in the Striatum

The adenosine A(2A) receptor (A(2A)R) is increasingly recognized as a novel therapeutic target in Parkinson disease. In striatopallidal neurons, the G-protein α(olf) subtype is required to couple this receptor to adenylyl cyclase activation. It is now well established that the βγ dimer also performs an active role in this signal transduction process. In principal, sixty distinct βγ dimers could arise from combinatorial association of the five known β and 12 γ subunit genes. However, key questions regarding which βγ subunit combinations exist and whether they perform specific signaling roles in the context of the organism remain to be answered. To explore these questions, we used a gene targeting approach to specifically ablate the G-protein γ(7) subtype. Revealing a potentially new signaling paradigm, we show that the level of the γ(7) protein controls the hierarchial assembly of a specific G-protein α(olf)β(2)γ(7) heterotrimer in the striatum. Providing a probable basis for the selectivity of receptor signaling, we further demonstrate that loss of this specific G-protein heterotrimer leads to reduced A(2A)R activation of adenylyl cyclase. Finally, substantiating an important role for this signaling pathway in pyschostimulant responsiveness, we show that mice lacking the G-protein γ(7) subtype exhibit an attenuated behavioral response to caffeine. Collectively, these results further support the A(2A)R G-protein α(olf)β(2)γ(7) interface as a possible therapeutic target for Parkinson disease.

Requirement of open headpiece conformation for activation of leukocyte integrin αXβ2

Negative stain electron microscopy (EM) and adhesion assays show that alpha(X)beta(2) integrin activation requires headpiece opening as well as extension. An extension-inducing Fab to the beta(2) leg, in combination with representative activating and inhibitory Fabs, were examined for effect on the equilibrium between the open and closed headpiece conformations. The two activating Fabs stabilized the open headpiece conformation. Conversely, two different inhibitory Fabs stabilized the closed headpiece conformation. Adhesion assays revealed that alpha(X)beta(2) in the extended-open headpiece conformation had high affinity for ligand, whereas both the bent conformation and the extended-closed headpiece conformation represented the low affinity state. Intermediate integrin affinity appears to result not from a single conformational state, but from a mixture of equilibrating conformational states.

Regional distribution of high and low affinity states of dopamine D2/3 receptor binding in humans: A PET study using [11C]MNPA and [11C]raclopride

Regional distribution of high and low affinity states of dopamine D2/3 receptor binding in humans: A PET study using [11C]MNPA and [11C]raclopride

Role of H 2O 2 on the kinetics of low-affinity high-capacity Na +-dependent alanine transport in SHR proximal tubular epithelial cells

The presence of high and low sodium affinity states for the Na +-dependent [ 14C]- l-alanine uptake in immortalized renal proximal tubular epithelial (PTE) cells was previously reported (Am. J. Physiol. 293 (2007) R538–R547). This study evaluated the role of H 2O 2 on the Na +-dependent [ 14C]- l-alanine uptake of ASCT2 in immortalized renal PTE cells from Wistar Kyoto rat (WKY) and spontaneously hypertensive rat (SHR). Na + dependence of [ 14C]- l-alanine uptake was investigated replacing NaCl with an equimolar concentration of choline chloride in vehicle- and apocynin-treated cells. Na + removal from the uptake solution abolished transport activity in both WKY and SHR PTE cells. Decreases in H 2O 2 levels in the extracellular medium significantly reduced Na +- K m and V max values of the low-affinity high-capacity component in SHR PTE cells, with no effect on the high-affinity low-capacity state of the Na +-dependent [ 14C]- l-alanine uptake. After removal of apocynin from the culture medium, H 2O 2 levels returned to basal values within 1 to 3 h in both WKY and SHR PTE cells and these were found stable for the next 24 h. Under these experimental conditions, the Na +- K m and V max of the high-affinity low-capacity state were unaffected and the low-affinity high-capacity component remained significantly decreased 1 day but not 4 days after apocynin removal. In conclusion, H 2O 2 in excess is required for the presence of a low-affinity high-capacity component for the Na +-dependent [ 14C]- l-alanine uptake in SHR PTE cells only. It is suggested that Na + binding in renal ASCT2 may be regulated by ROS in SHR PTE cells.

Human T Lymphocyte Isolation, Culture and Analysis of Migration <em>In Vitro</em>

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 μm/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration <em>In Vitro</em>

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 μm/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Two-State Conformations in the Hyaluronan-Binding Domain Regulate CD44 Adhesiveness under Flow Condition

The hyaluronan (HA) receptor CD44 mediates cell adhesion in leukocyte trafficking and tumor metastasis. Our previous nuclear magnetic resonance (NMR) studies revealed that the CD44 hyaluronan-binding domain (HABD) alters its conformation upon HA binding, from the ordered (O) to the partially disordered (PD) conformation. Here, we demonstrate that the HABD undergoes an equilibrium between the O and PD conformations, in either the presence or absence of HA, which explains the seemingly contradictory X-ray and NMR structures of the HA-bound HABD. An HABD mutant that exclusively adopts the PD conformation displayed a higher HA affinity than the wild-type. Rolling of the cells expressing the mutant CD44 was less efficient than those expressing the wild-type, due to the decreased tether frequency and the slow cellular off rate. Considering that the mutant CD44, devoid of the low-affinity state, exhibited impaired rolling, we conclude that the coexistence of the high- and low-affinity states of the HABD is essential for the CD44-mediated rolling.

A Low Affinity Ground State Conformation for the Dynein Microtubule Binding Domain

Dynein interacts with microtubules through a dedicated binding domain that is dynamically controlled to achieve high or low affinity, depending on the state of nucleotide bound in a distant catalytic pocket. The active sites for microtubule binding and ATP hydrolysis communicate via conformational changes transduced through a approximately 10-nm length antiparallel coiled-coil stalk, which connects the binding domain to the roughly 300-kDa motor core. Recently, an x-ray structure of the murine cytoplasmic dynein microtubule binding domain (MTBD) in a weak affinity conformation was published, containing a covalently constrained beta(+) registry for the coiled-coil stalk segment (Carter, A. P., Garbarino, J. E., Wilson-Kubalek, E. M., Shipley, W. E., Cho, C., Milligan, R. A., Vale, R. D., and Gibbons, I. R. (2008) Science 322, 1691-1695). We here present an NMR analysis of the isolated MTBD from Dictyostelium discoideum that demonstrates the coiled-coil beta(+) registry corresponds to the low energy conformation for this functional region of dynein. Addition of sequence encoding roughly half of the coiled-coil stalk proximal to the binding tip results in a decreased affinity of the MTBD for microtubules. In contrast, addition of the complete coiled-coil sequence drives the MTBD to the conformationally unstable, high affinity binding state. These results suggest a thermodynamic coupling between conformational free energy differences in the alpha and beta(+) registries of the coiled-coil stalk that acts as a switch between high and low affinity conformations of the MTBD. A balancing of opposing conformations in the stalk and MTBD enables potentially modest long-range interactions arising from ATP binding in the motor core to induce a relaxation of the MTBD into the stable low affinity state.

Structural Basis for Mechanical Force Regulation of the Adhesin FimH via Finger Trap-like β Sheet Twisting

The Escherichia coli fimbrial adhesive protein, FimH, mediates shear-dependent binding to mannosylated surfaces via force-enhanced allosteric catch bonds, but the underlying structural mechanism was previously unknown. Here we present the crystal structure of FimH incorporated into the multiprotein fimbrial tip, where the anchoring (pilin) domain of FimH interacts with the mannose-binding (lectin) domain and causes a twist in the beta sandwich fold of the latter. This loosens the mannose-binding pocket on the opposite end of the lectin domain, resulting in an inactive low-affinity state of the adhesin. The autoinhibition effect of the pilin domain is removed by application of tensile force across the bond, which separates the domains and causes the lectin domain to untwist and clamp tightly around the ligand like a finger-trap toy. Thus, beta sandwich domains, which are common in multidomain proteins exposed to tensile force in vivo, can undergo drastic allosteric changes and be subjected to mechanical regulation.

D2 receptor occupancy in conscious rat brain is not significantly distinguished with [3H]‐MNPA, [3H]‐(+)‐PHNO, and [3H]‐raclopride

Positron emission tomography (PET) antagonist ligands such as [(11)C]-raclopride are commonly used to study dopamine D2 receptor (D2) binding of antipsychotics. It has been suggested that agonist radioligands bind preferentially to the high-affinity state of D2 receptor and may provide a more relevant means of assessing D2 occupancy. The main objective of this study was to determine if D2 receptor occupancy (RO) could be differentiated with agonist and antagonist radioligands in vivo. Agonist radioligands [(3)H]-MNPA and [(3)H]-(+)-PHNO were synthesized and compared to antagonist [(3)H]-raclopride in the in vitro binding and in vivo occupancy studies. In vivo, unanesthetized rats were pretreated with quinpirole (full agonist), aripiprazole (partial agonist), or haloperidol (antagonist) prior to administration of the agonist or antagonist radioligand. All three pretreatment compounds showed equivalent dose-dependent D2 receptor occupancy in the rat striatum with each radioligand. The in vivo receptor occupancy results suggested that the binding of quinpirole, aripiprazole, and haloperidol to the high or low affinity state of the D2 receptor could not be differentiated using radiolabeled agonists or antagonists, presumably due to a predominance of high affinity states of the D2 receptor in vivo. This hypothesis was supported in part by the in vitro binding results. Our in vitro results show that [(3)H]-MNPA binds to D2S transfected CHO cell membranes at a single high affinity site. Displacement of [(3)H]-(+)-PHNO binding by quinpirole and elimination of most [(3)H]-(+)-PHNO binding by the guanine nucleotide GppNHp in striatal membranes suggest that the majority of D2 in striatal tissue is G-protein coupled. Together, these findings suggest that D2 agonist radioligands produce in vivo receptor occupancy comparable to [(3)H]-raclopride.

Dopamine β‐hydroxylase‐deficient mice have normal densities of D2 dopamine receptors in the high‐affinity state based on in vivo PET imaging and in vitro radioligand binding

In vitro, D(2) dopamine receptors (DAR) can exist in low- and high-affinity states for agonists and increases of D(2) receptors in high-affinity state have been proposed to underlie DA receptor supersensitivity in vivo. Deletion of the gene for dopamine beta-hydroxylase (DBH) causes mice to become hypersensitive to the effects of psychostimulants, and in vitro radioligand binding results suggest an increased percentage of D(2) receptors in a high-affinity state. To determine whether DBH knockout mice display an increase of high-affinity state D(2) receptors in vivo, we scanned DBH knockout and control mice with the agonist PET radioligand [(11)C]MNPA, which is thought to bind preferentially to the high-affinity state of the D(2) receptor. In addition, we performed in vitro binding experiments on striatal homogenates with [(3)H]methylspiperone to measure B(max) values and the percentages of high- and low-affinity states of the D(2) receptor. We found that the in vivo striatal binding of [(11)C]MNPA was similar in DBH knockout mice and heterozygous controls and the in vitro B(max) values and percentages of D(2) receptors in the high-affinity state, were not significantly different between these two groups. In summary, our results suggest that DBH knockout mice have normal levels of D(2) receptors in the high-affinity state and that additional mechanisms contribute to their behavioral sensitivity to psychostimulants.

Excited protein states of human tear lipocalin for low- and high-affinity ligand binding revealed by functional AB loop motion

Human tear lipocalin (TL), a prominent member of lipocalin family, exhibits functional and structural promiscuity. The plasticity of loop regions modulates entry to the ligand pocket at the “open” end of the eight-stranded β-barrel. Site-directed multi-distance measurements using fluorescence resonance energy transfer between functional loops register two excited protein states for low- and high-affinity ligand binding. At low pH, the longest loop AB adopts the conformation of the low-affinity excited protein state that matches the crystal structure of holo-TL at pH 8. A “crankshaft” like movement is detected for the loop AB in a low pH transition. At pH 7.3 the holo-protein assumes a high-affinity excited protein state, in which the loop AB is more compact (RMS = 3.1 Å). In the apo-holo transition, the reporter Trp 28 moves about 4.5 Å that reflects a decrease in distance between Glu27 and Lys108. This interaction fixes the loop AB conformation for the high-affinity mode. No such movement is detected at low pH, where Glu27 is protonated. Data strongly indicate that the protonation state of Glu27 modulates the conformation of the loop AB for high- and low-affinity binding.

Determination of the intracellular aqueous/lipid interface of integrin alpha 2b in vivo

Integrins are a family of cell adhesion receptors that mediate cell migration, extracellular matrix assembly and platelet aggregation. They are heterodimeric proteins composed of alpha and beta subunits. Integrins exist in a default low affinity state in the cell membrane. The highly conserved intracellular memebrane proximal region of the alpha subunit is considered to play a critical role in constraining the receptor in the low affinity state, however, the location of this region in the cellular environment remains controversial. In this study, we explored the location of the membrane proximal region of integrin alpha 2b by substituted cysteine accessibility method (SCAM). Residues in the membrane proximal region of integrin alpha 2b were individually substituted with cysteines and subjected to in situ chemical probing with a cysteine specific reagent, biotin maleimide. Results showed that Lys‐994 marks the first aqueous exposed residue in the region, consistent with the in vitro findings. Further functional analysis with a flow cytometry assay revealed that all the mutant receptors were active. On the basis of the results, we conclude that the in vitro determined aqueous/lipid interface of integrin alpha 2b represents the active state of the receptor.Source of Research Support: UCLA Nephrology Research Fund

Dopamine D2 Receptors as Treatment Targets in Schizophrenia

The antipsychotic effectiveness of chlorpromazine and haloperidol started a search for their therapeutic targets. The antipsychotic receptor target turned out to be a dopamine receptor, now cloned as the dopamine D2 receptor. The D2 receptor is the common target for antipsychotics. Antipsychotic clinical doses correlate with their affinities for this receptor. Therapeutic doses of antipsychotics occupy 60 to 80% of brain D2 receptors in patients, but aripiprazole occupies up to 90%. While antipsychotics may take up to six hours to occupy D2 receptors, much clinical improvement occurs within a few days. The receptor has high- and low-affinity states. The D2High state is functional for dopamine-like agonists such as aripiprazole. Most individuals with schizophrenia are supersensitive to dopamine. Animal models of psychosis show that a variety of risk factors, genetic and nongenetic, are associated with behavioral supersensitivity to dopamine, reflected in elevated levels of dopamine D2High receptors. Although antipsychotics such as haloperidol alleviate psychosis and reverse the elevation of D2High receptors, long-term use of traditional antipsychotics can further enhance dopamine supersensitivity in patients. Therefore, switching from a traditional antipsychotic to an agonist antipsychotic such as aripiprazole can result in the emergence of psychotic signs and symptoms. Clozapine and quetiapine do not elicit parkinsonism and rarely result in tardive dyskinesia because they are released from D2 within 12 to 24 hours. Traditional antipsychotics remain attached to D2 receptors for days, preventing relapse, but allowing accumulation that can lead to tardive dyskinesia. Future goals include imaging D2High receptors and desensitizing them in early-stage psychosis.

Shear‐enhanced binding of intestinal colonization factor antigen I of enterotoxigenic Escherichia coli

In the intestine, enterotoxigenic Escherichia coli works against peristaltic forces, adhering to the epithelium via the colonization factor antigen I (CFA/I) fimbrial adhesin CfaE. The CfaE adhesin is similar in localization and tertiary (but not primary) structure to FimH, the type 1 fimbrial adhesin of uropathogenic E. coli, which shows shear-dependent binding to epithelial receptors by an allosteric catch-bond mechanism. Thus, we speculated that CfaE is also capable of shear-enhanced binding. Indeed, bovine erythrocytes coursing over immobilized CFA/I fimbriae in flow chambers exhibited low accumulation levels and fast rolling at low shear, but an 80-fold increase in accumulation and threefold decrease in rolling velocity at elevated shear. This effect was reversible and abolished by pre-incubation of fimbriae with anti-CfaE antibody. Erythrocytes bound to whole CfaE in the same shear-enhanced manner, but to CfaE adhesin domain in a shear-inhibitable fashion. Residue replacements designed to disrupt CfaE interdomain interaction decreased the shear dependency of adhesion and increased binding under static conditions to human intestinal epithelial cells. These findings indicate that close interaction between adhesive and anchoring pilin domains of CfaE keeps the former in a low-affinity state that toggles into a high-affinity state upon separation of two domains, all consistent with an allosteric catch-bond mechanism of CfaE binding.

Distinctive Properties of the Hyaluronan-binding Domain in the Lymphatic Endothelial Receptor Lyve-1 and Their Implications for Receptor Function

The lymphatic endothelial hyaluronan (HA) receptor Lyve-1 is a member of the Link protein superfamily most similar to the leukocyte HA receptor CD44. However, the structure of Lyve-1 and the nature of its interaction with ligand are obscure. Here we present new evidence that Lyve-1 is functionally distinct from CD44. Using truncation mutagenesis we confirm that Lyve-1 in common with CD44 contains an extended HA-binding unit, comprising elements flanking the N and C termini of the consensus lectin-like Link module, bridged by a third conserved disulfide linkage that is critical for HA binding. In addition, we identify six essential residues Tyr-87, Ile-97, Arg-99, Asn-103, Lys-105, and Lys-108 that define a compact HA-binding surface on Lyve-1, encompassing the epitope for an adhesion-blocking monoclonal antibody 3A, in an analogous position to the HA-binding surface in CD44. The overtly electrostatic character of HA binding in Lyve-1 and its sensitivity to ionic strength (IC(50) of 150 mm NaCl) contrast markedly with CD44 (IC(50) > 2 m NaCl) in which HA binding is mediated by hydrogen bonding and hydrophobic interactions. In addition, unlike the extended Link module in CD44, which binds HA efficiently when expressed as a soluble monomer (K(d) = 65.7 mum), that of Lyve-1 requires artificial dimerization, although the full ectodomain is active as a monomer (K(d) = 35.6 mum). Finally, full-length Lyve-1 did not form stable dimers in binding-competent 293T transfectants when assessed using bioluminescent resonance energy transfer. These results reveal that elements additional to the extended Link module are required to stabilize HA binding in Lyve-1 and indicate important structural and functional differences with CD44.