Mechanical modulation of receptor-ligand interactions at cell-cell interfaces

Cell surface receptors have been extensively studied because they initiate and regulate signal transduction cascades leading to a variety of functional cellular outcomes. An important class of immune receptors (e.g., T-cell antigen receptors) whose ligands are anchored to the surfaces of other cells remain poorly understood. The mechanism by which ligand binding initiates receptor phosphorylation, a process termed “receptor triggering”, remains controversial. Recently, direct measurements of the (two-dimensional) receptor-ligand complex lifetimes at cell-cell interface were found to be smaller than (three-dimensional) lifetimes in solution but the underlying mechanism is unknown. At the cell-cell interface, the receptor-ligand complex spans a short intermembrane distance (15 nm) compared to long surface molecules (LSMs) whose ectodomains span >40 nm and these LSMs include phosphatases (e.g., CD45) that dephosphorylate the receptor. It has been proposed that size-based segregation of LSMs from a receptor-ligand complex is a mechanism of receptor triggering but it is unclear whether the mechanochemistry supports such small-scale segregation. Here we present a nanometer-scale mathematical model that couples membrane elasticity with the compressional stiffness and lateral mobility of LSMs. We find robust supradiffusive segregation of LSMs from a single receptor-ligand complex. The model predicts that LSM redistribution will result in a time-dependent tension on the complex leading to a decreased two-dimensional lifetime. Interestingly, the model predicts a nonlinear relationship between the three- and two-dimensional lifetimes, which can enhance the ability of receptors to discriminate between similar ligands.     

Monitoring receptor-ligand interactions between surfaces by thermal fluctuations

We describe a new method for determining receptor-ligand association/dissociation events across the interface of two surfaces (two-dimensional binding) by monitoring abrupt decrease/resumption in thermal fluctuations of a biomembrane force probe. Our method has been validated by rigorous control experiments and kinetic experiments. We show that cellular on-rate of association can be measured by analysis of intervals from a dissociation event to the next association event (waiting times). Similarly, off-rate of molecular dissociation can be measured by analysis of intervals from an association event to the next dissociation event (bond lifetimes). Different types of molecular bonds could be distinguished by different levels of reduction in thermal fluctuations. This novel method provides a powerful tool to study cell adhesion and signaling mediated by single or multiple receptor-ligand species.     

Real time non-invasive imaging of receptor-ligand interactions in vivo

Non-invasive longitudinal detection and evaluation of gene expression in living animals can provide investigators with an understanding of the ontogeny of a gene's biological function(s). Currently, mouse model systems are used to optimize magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), and optical imaging modalities to detect gene expression and protein function. These molecular imaging strategies are being developed to assess tumor growth and the tumor microenvironment. In addition, pre-labeling of progenitor cells can provide invaluable information about the developmental lineage of stem cells both in organogenesis and tumorigenesis. The feasibility of this approach has been extensively tested by targeting of endogenous tumor cell receptors with labeled ligand (or ligand analog) reporters and targeting enzymes with labeled substrate (or substrate analog). We will primarily discuss MRI, PET, and SPECT imaging of cell surface receptors and the feasibility of non-invasive imaging of gene expression using the tumor microenvironment (e.g., hypoxia) as a conditional regulator of gene expression.     

Thermodynamic descriptors,profiles and driving forces in membrane receptor-ligand interactions

Extension of the (isothermal) Gibbs–Helmholtz equation for the heat capacity terms (ΔC_p) allows formulating a temperature function of the free (Gibbs) energy change (ΔG). An approximation of the virtually unknown ΔC_p temperature function enables then to determine and numerically solve temperature functions of thermodynamic parameters ΔH and ΔS (enthalpy and entropy change, respectively). Analytical solutions and respective numeric procedures for several such approximation formulas are suggested in the presented paper. Agreement between results obtained by this analysis with direct microcalorimetric measurements of ΔH (and ΔC_p derived from them) was approved on selected cases of biochemical interactions presented in the literature. Analysis of several ligand-membrane receptor systems indicates that temperature profiles of ΔH and ΔS are parallel, largely not monotonic, and frequently attain both positive and negative values within the current temperature range of biochemical reactions. Their course is determined by the reaction change of heat capacity: temperature extremes (maximum or minimum) of both ΔH and ΔS occur at ΔC_p?=?0, for most of these systems at roughly 285–305 K. Thus, the driving forces of these interactions may change from enthalpy-, entropy-, or enthalpy-entropy-driven in a narrow temperature interval. In contrast, thermodynamic parameters of ligand-macromolecule interactions in solutions (not bound to a membrane) mostly display a monotonic course. In the case of membrane receptors, thermodynamic discrimination between pharmacologically defined groups—agonists, partial agonists, antagonists—is in general not specified and can be achieved, in the best, solely within single receptor groups.     

Toxic ligand conjugates as tools in the study of receptor-ligand interactions

We have constructed hybrid proteins in which the toxic A chains of ricin or diptheria toxin have been linked to either asialofetuin, fetuin, or epidermal growth factor (EGF). Both ASF-RTA and ASF-DTA are potent toxins on cultured rat hepatocytes, cells that display the asialoglycoprotein receptor. Toxicity of these two compounds is restricted to hepatocytes and can be blocked by asialoglycoproteins but not the native glycoproteins or asialoagalactoglycoprotein derivatives, indicating that the toxicity of the conjugates is mediated by the hepatic asialoglycoprotein receptor. The EGF-RTA conjugate is an extremely potent toxin on cells that can bind the hormone, but is only poorly effective on cells that are unable to bind EGF. The EGF-DTA conjugate, in contrast, is unable to kill 3T3 cells and is at least two orders of magnitude less effective than EGF-RTA on A431 cells, a cell line with 1-2 X 10(6) EGF receptors per cell. However, when EGF-RTA and EGF-DTA were tested on primary liver hepatocyte cultures, which were susceptible to both ASF-RTA and ASF-DTA, both EGF conjugates were potent toxins. Sensitivity of the hepatocyte cultures to ricin toxicity increases slightly during a 52-hr culture period. In contrast, sensitivity to EGF-RTA and ASF-RTA decline dramatically during this period. Receptors for both ligands remain plentiful on the cell surface during this time.     

Thermodynamic descriptors, profiles and driving forces in membrane receptor-ligand interactions

Extension of the (isothermal) Gibbs-Helmholtz equation for the heat capacity terms (ΔC(p)) allows formulating a temperature function of the free (Gibbs) energy change (ΔG). An approximation of the virtually unknown ΔC(p) temperature function enables then to determine and numerically solve temperature functions of thermodynamic parameters ΔH and ΔS (enthalpy and entropy change, respectively). Analytical solutions and respective numeric procedures for several such approximation formulas are suggested in the presented paper. Agreement between results obtained by this analysis with direct microcalorimetric measurements of ΔH (and ΔC(p) derived from them) was approved on selected cases of biochemical interactions presented in the literature. Analysis of several ligand-membrane receptor systems indicates that temperature profiles of ΔH and ΔS are parallel, largely not monotonic, and frequently attain both positive and negative values within the current temperature range of biochemical reactions. Their course is determined by the reaction change of heat capacity: temperature extremes (maximum or minimum) of both ΔH and ΔS occur at ΔC(p)=0, for most of these systems at roughly 285-305 K. Thus, the driving forces of these interactions may change from enthalpy-, entropy-, or enthalpy-entropy-driven in a narrow temperature interval. In contrast, thermodynamic parameters of ligand-macromolecule interactions in solutions (not bound to a membrane) mostly display a monotonic course. In the case of membrane receptors, thermodynamic discrimination between pharmacologically defined groups-agonists, partial agonists, antagonists-is in general not specified and can be achieved, in the best, solely within single receptor groups.     

Dissecting the chemistry of nicotinic receptor-ligand interactions with infrared difference spectroscopy.

The physical interactions that occur between the nicotinic acetylcholine receptor from Torpedo and the agonists carbamylcholine and tetramethylamine have been studied using both conventional infrared difference spectroscopy and a novel double-ligand difference technique. The latter was developed to isolate vibrational bands from residues in a membrane receptor that interact with individual functional groups on a small molecule ligand. The binding of either agonist leads to an increase in vibrational intensity at frequencies centered near 1663, 1655, 1547, 1430, and 1059 cm(-1) indicating that both induce a conformational change from the resting to the desensitized state. Vibrational shifts near 1580, 1516, 1455, 1334, and between 1300 and 1400 cm(-1) are assigned to structural perturbations of tyrosine and possibly both tryptophan and charged carboxylic acid residues upon the formation of receptor-quaternary amine interactions, with the relatively intense feature near 1516 cm(-1) indicating a key role for tyrosine. Other vibrational bands suggest the involvement of additional side chains in agonist binding. Two side-chain vibrational shifts from 1668 and 1605 cm(-1) to 1690 and 1620 cm(-1), respectively, could reflect the formation of a hydrogen bond between the ester carbonyl of carbamylcholine and an arginine residue. The results demonstrate the potential of the double-ligand difference technique for dissecting the chemistry of membrane receptor-ligand interactions and provide new insight into the nature of nicotinic receptor-agonist interactions.     

Impact of N-terminal domains for corticotropin-releasing factor (CRF) receptor-ligand interactions

The large extracellular N-terminal domains (NTs) of class B G protein-coupled receptors serve as major ligand binding sites. However, little is known about the ligand requirements for interactions with these receptor domains. Recently, we have shown that the most potent CRF receptor agonist urocortin 1 (Ucn1) has two segregated receptor binding sites Ucn1(1-21) and Ucn1(32-40). For locating the receptor domains interacting with these two sites, we have investigated the binding of appropriate Ucn1 analogues to the receptor N-termini compared to the corresponding full-length receptors. For this purpose receptor NTs of CRF(rat) subtypes 1 and 2(alpha) without their signal sequences were overexpressed in Escherichia coli and folded in vitro. For CRF2(a)-rNT, which bears five cysteine residues (C2-C6), the disulfide arrangement C2-C5 and C4-C6 was found, leaving C3 free. This is consistent with the disulfide pattern of CRF1-rNT, which has six cysteines and in which C1 is paired with C3. Binding studies of N-terminally truncated or C-terminally modified Ucn1 analogues demonstrate that it is the C-terminal part, Ucn1(11-40), that binds to receptor NT, indicating a two-domain binding mechanism for Ucn binding to receptor NT. Since the binding of Ucn1 to the juxtamembrane domain has been shown to be segregated from binding to the receptor N-terminus [Hoare et al. (2004) Biochemistry 43, 3996-4011], a third binding domain should exist, probably comprising residues 8-10 of Ucn, which particularly contribute to a high-affinity binding to full-length receptors but not to receptor NT.     

Beyond induced-fit receptor-ligand interactions: structural changes that can significantly extend bond lifetimes

While the lifetime of conventional receptor-ligand interactions is shortened by tensile mechanical force, some recently discovered interactions, termed catch bonds, can be strengthened by force. Motivated by the search for the underpinning structural mechanisms, we here explore the structural dynamics of the binding site of the bacterial adhesive protein FimH by molecular dynamics and steered molecular dynamics. While the crystal structure of only one FimH conformation has been reported so far, we describe two distinctively different conformations of the mannose-bound FimH binding site. Force-induced dissociation was slowed when the mannose ring rotated such that additional force-bearing hydrogen bonds formed with the base of the FimH binding pocket. The lifetime of the complex was further enhanced significantly by rigidifying this base. We finally show how even sub-angstrom spatial alterations of the hydrogen bonding pattern within the base can lead to significantly decreased bond lifetimes.     

Roles of cell and microvillus deformation and receptor-ligand binding kinetics in cell rolling

Polymorphonuclear leukocyte (PMN) recruitment to sites of inflammation is initiated by selectin-mediated PMN tethering and rolling on activated endothelium under flow. Cell rolling is modulated by bulk cell deformation (mesoscale), microvillus deformability (microscale), and receptor-ligand binding kinetics (nanoscale). Selectin-ligand bonds exhibit a catch-slip bond behavior, and their dissociation is governed not only by the force but also by the force history. Whereas previous theoretical models have studied the significance of these three "length scales" in isolation, how their interplay affects cell rolling has yet to be resolved. We therefore developed a three-dimensional computational model that integrates the aforementioned length scales to delineate their relative contributions to PMN rolling. Our simulations predict that the catch-slip bond behavior and to a lesser extent bulk cell deformation are responsible for the shear threshold phenomenon. Cells bearing deformable rather than rigid microvilli roll slower only at high P-selectin site densities and elevated levels of shear (>or=400 s(-1)). The more compliant cells (membrane stiffness=1.2 dyn/cm) rolled slower than cells with a membrane stiffness of 3.0 dyn/cm at shear rates >50 s(-1). In summary, our model demonstrates that cell rolling over a ligand-coated surface is a highly coordinated process characterized by a complex interplay between forces acting on three distinct length scales.     

A model for the progression of receptor-ligand interactions during erythrocyte invasion by Plasmodium falciparum

Multiple and seemingly sequential interactions between parasite ligands and their receptors on host erythrocytes are an essential precursor to invasion by the obligate intracellular pathogen, Plasmodium falciparum. Consequently, identification and characterisation of the specific effectors that facilitate these recognition events are of special interest for the development of novel therapeutic and prophylactic solutions to malaria. There have been many recent advances regarding the identification of host-parasite receptor-ligand pairs, however the precise function and temporal aspects of these interactions are far from resolved. This review provides an update on the current details of these interactions to place them in sequence and super impose them upon the known kinetic events of invasion.     

Identification of a putative receptor-ligand pair controlling cell separation in plants

Cell separation events are important throughout the lifespan of a plant. To assure that the plant''s integrity is not compromised, such events, which depend on cell wall degradation, have to be tightly controlled both in time and space. The final step of floral organ abscission in Arabidopsis is controlled by INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), in that mutation of IDA causes a block in abscission. Overexpression results in early abscission of floral organs. In a recent article we show that this is also the case when overexpressing the related IDA-LIKE (IDL) proteins, indicating a degree of functional redundancy. Based on gene swap and deletion constructs introduced in the ida mutant and synthetic peptide assays we demonstrated that the conserved C-terminal motif (EPIP) of IDA and IDL1 was sufficient to replace IDA function. This function is dependent on the presence of the receptor-like kinases (RLK) HAESA (HAE) and HAESA-LIKE2 (HSL2), suggesting that an IDA peptide acts as a ligand interacting with these receptors. Our study further revealed that the five IDL genes are expressed at various sites where cell separation takes place. We suggest that the IDL proteins constitute a family of ligands that act through RLKs similar to HAESA and control cell separation at different sites and development stages during the life of the plant.Key words: Arabidopsis, signaling, receptor, ligand, gene-family, cell separation, HAE, IDA     

Leishmania amazonensis: multiple receptor-ligand interactions are involved in amastigote infection of human dendritic cells

In their mammalian hosts, Leishmania are obligate intracellular parasites that reside in macrophages and dendritic cells (DCs). In the present study, we have investigated in vitro the mechanisms of entry into human DCs of Leishmania amazonensis amastigotes isolated from lesions in nude mice (Am nude). The DC infection rate with Am nude was approximately 36%, while opsonization of Am nude with normal human serum and infected human serum increased the DC infection rates to 60% and 62%, respectively. Heat inactivation and depletion of antibodies in sera brought the DC infection rate down to 40%. The DC infection rate was inhibited after pre-treatment of Am nude with heparin. We were unable to implicate mannose-fucose receptors in the uptake of Am nude by DCs. Our data suggest that the ability of L. amazonensis amastigotes to infect human DCs involves the participation of at least three multiple receptor-ligand interactions, antibodies/FcR, complement components/CR and proteoglycans/heparin-binding protein.     

Estimation of receptor-ligand interactions by the use of a two-marker system in affinity capillary electrophoresis

The study of receptor-ligand interactions by affinity capillary electrophoresis (ACE) requires an accurate form of analysis. Here, we examine the use of two noninteracting standards (markers) in the analysis of binding constant data in ACE studies. This concept is demonstrated using two model systems: carbonic anhydrase B (CAB, EC 4.2.1.1) and arylsulfonamides, and vancomycin (Van) from Streptomyces orientalis and the dipeptide N-acetyl-d-Ala-d-Ala. In this procedure a plug of receptor and noninteracting standards is injected, and analysis of the change in the relative migration time ratio of the receptor, relative to the noninteracting standards, as a function of the concentration of the ligand yields a value for the binding constant. The findings described here demonstrate that data from ACE studies can best be analyzed using two noninteracting standards, yielding values comparable to those estimated using other binding and ACE techniques.     

Natural killer (NK)-like cytotoxic activity of allospecific T cell receptor-gamma,delta+ T cell clones. Distinct receptor-ligand interactions mediate NK-like and allospecific cytotoxicity

We recently generated a series of human alloantigen-specific, CD3+,TCR-gamma,delta+ clones by stimulating CD3+,CD4-,CD8- T cells from normal individuals with allogeneic lymphoblastoid cell lines (LCL). As reported previously, these clones display cytotoxic activity against their specific stimulators but not against irrelevant LCL. Further studies of these and other TCR-gamma,delta+ clones, described in this report, indicate that most but not all of these clones express the NK cell associated marker, NKH-1 or Leu-19, and kill NK-sensitive targets such as the K562 and Molt 4 lines, but not an irrelevant LCL or NK-resistant line, Raji. TCR-gamma,delta+ clones which lacked expression of Leu-19 lysed their allospecific targets but had no detectable NK activity. The allospecific cytotoxicity of Leu-19+ and Leu-19- clones was inhibited by mAb to CD3 or the TCR delta-chain. In contrast, the NK-like activity of the Leu-19+ clones was enhanced by these antibodies over a wide range of antibody concentration. Although mAb to LFA-1 markedly inhibited both the allospecific and NK-like activity of these clones, an HLA class I framework specific mAb (W6/32) had no effect on NK-like cytolysis but did inhibit allospecific killing, suggesting that the target structures on the surface of allospecific and NK-sensitive cells are distinct. The receptors utilized by the TCR-gamma,delta+ clones to recognize NK-sensitive and allospecific targets are also distinct, since killing of NK-sensitive targets was blocked by the presence of cold (unlabeled) NK-sensitive cells but not by cold allospecific targets, whereas allospecific cytolysis was inhibited by cold allospecific targets but not by NK-sensitive cells. We conclude that some CD3+,TCR-gamma,delta+ clones exhibit NK-like as well as allospecific killing and that these two activities are mediated by distinct receptor-ligand interactions.     

Effect of receptor-ligand affinity on the strength of endothelial cell adhesion.

The objective of this study was to determine the effect of receptor-ligand affinity on the strength of endothelial cell adhesion. Linear and cyclic forms of the fibronectin (Fn) cell-binding domain peptide Arg-Gly-Asp (RGD) were covalently immobilized to glass, and Fn was adsorbed onto glass slides. Bovine aortic endothelial cells attached to the surfaces for 15 min. The critical wall shear stress at which 50% of the cells detached increased nonlinearly with ligand density and was greater with immobilized cyclic RGD than with immobilized linear RGD or adsorbed Fn. To directly compare results for the different ligand densities, the receptor-ligand dissociation constant and force per bond were estimated from data for the critical shear stress and contact area. Total internal reflection fluorescence microscopy was used to measure the contact area as a function of separation distance. Contact area increased with increasing ligand density. Contact areas were similar for the immobilized peptides but were greater on surfaces with adsorbed Fn. The dissociation constant was determined by nonlinear regression of the net force on the cells to models that assumed that bonds were either uniformly stressed or that only bonds on the periphery of the contact region were stressed (peeling model). Both models provided equally good fits for cells attached to immobilized peptides whereas the peeling model produced a better fit of data for cells attached to adsorbed Fn. Cyclic RGD and linear RGD both bind to the integrin alpha v beta 3, but immobilized cyclic RGD exhibited a greater affinity than did linear RGD. Receptor affinities of Fn adsorbed to glycophase glass and Fn adsorbed to glass were similar. The number of bonds was calculated assuming binding equilibrium. The peeling model produced good linear fits between bond force and number of bonds. Results of this study indicate that 1) bovine aortic endothelial cells are more adherent on immobilized cyclic RGD peptide than linear RGD or adsorbed Fn, 2) increased adhesion is due to a greater affinity between cyclic RGD and its receptor, and 3) the affinity of RGD peptides and adsorbed Fn for their receptors is increased after immobilization.