Hormone receptors.

Hormone receptors are proteins located on the cell membrane or in the cell cytoplasm. Their function is to recognize and bind the respective hormone; the biological action of the hormone originates from the hormone-receptor complex. Specific receptors have been found for all known hormones. Monitoring the equilibration between hormone, receptor, and hormone-receptor complex (“determination of receptor binding”) permits quantitative determinations of hormones, allows the characterization of endocrinal disturbances, and contributes to the elucidation of structure-affinity relationships.     

Monitoring the dynamics of ligand-receptor complexes on model membranes

Ligand-induced cross-linking of cell surface receptors is a basic paradigm of signal activation by many transmembrane receptors. After ligand binding, the receptor complexes formed on the membrane are dynamically maintained by two-dimensional protein-protein interactions on the membrane. The biophysical principles governing the dynamics of such interactions have not been understood, mainly because the measurement of lateral interactions on membranes so far has not been experimentally addressed. Here, we describe a generic approach for measuring two-dimensional dissociation rate constants in vitro using a novel high-affinity chelator lipid for reconstituting a ternary cytokine-receptor complex on solid-supported membranes. While monitoring the interaction between the ligand and one of the receptor subunits on the membrane by fluorescence resonance energy transfer, the equilibrium on the surface was perturbed by rapidly tethering a large excess of the unlabeled receptor subunit. Displacement of labeled by unlabeled protein in the ternary complex was detected as a recovery of the donor quenching. Since the dissociation of the ligand-receptor complex in plane of the membrane was the rate-limiting step under these conditions, the two-dimensional rate constant of this process was determined. Strikingly, the two-dimensional dissociation was much slower than ligand dissociation into solution, suggesting that membrane tethering significantly affects the dissociation process. This result highlights the importance of studying ligand-receptor complexes tethered to membranes for understanding the principles governing signal activation by ligand-induced receptor assembling.     

Analysis of Interactions between the Epidermal Growth Factor Receptor and Soluble Ligands on the Basis of Single‐Molecule Diffusivity in the Membrane of Living Cells

We present a single‐molecule diffusional‐mobility‐shift assay (smDIMSA) for analyzing the interactions between membrane and water‐soluble proteins in the crowded membrane of living cells. We found that ligand–receptor interactions decreased the diffusional mobility of ErbB receptors and β‐adrenergic receptors, as determined by single‐particle tracking with super‐resolution microscopy. The shift in diffusional mobility was sensitive to the size of the water‐soluble binders that ranged from a few tens of kilodaltons to several hundred kilodaltons. This technique was used to quantitatively analyze the dissociation constant and the cooperativity of antibody interactions with the epidermal growth factor receptor and its mutants. smDIMSA enables the quantitative investigation of previously undetected ligand–receptor interactions in the intact membrane of living cells on the basis of the diffusivity of single‐molecule membrane proteins without ligand labeling.     

Convection, diffusion and reaction in a surface-based biosensor: modeling of cooperativity and binding site competition on the surface and in the hydrogel

We study theoretically the transport and kinetic processes underlying the operation of a biosensor (particularly the surface plasmon sensor "Biacore") used to study the surface binding kinetics of biomolecules in solution to immobilized receptors. Unlike previous studies, we concentrate mainly on the modeling of system-specific phenomena rather than on the influence of mass transport limitations on the intrinsic kinetic rate constants determined from binding data. In the first problem, the case of two-site binding where each receptor unit on the surface can accommodate two analyte molecules on two different sites is considered. One analyte molecule always binds first to a specific site. Subsequently, the second analyte molecule can bind to the adjacent unoccupied site. In the second problem, two different analytes compete for one binding site on the same surface receptor. Finally, the third problem considers the case of positive cooperativity among bound molecules in the hydrogel using a simple mean-field approach. The transport in both the flow channel and the hydrogel phases of the biosensor is taken into account in this case (with few exceptions, most previous studies assume a simpler model in which the hydrogel is treated as a planar surface with the receptors). We consider simultaneously diffusion and convection through the flow channel together with diffusion and cooperativity binding on the surface and in the hydrogel. In each case, typical results for the concentration contours of the free and bound molecules in the flow channel and hydrogel regions are presented together with the time-dependent association/dissociation curves and reaction rates. For binding site competition, the analysis predicts overshoot phenomena.     

Modeling negative cooperativity in streptavidin adsorption onto biotinylated microtubules

The nanoscale architecture of binding sites can result in complex binding kinetics. Here, the adsorption of streptavidin and neutravidin to biotinylated microtubules is found to exhibit negative cooperativity due to electrostatic interactions and steric hindrance. This behavior is modeled by a newly developed kinetic analogue of the Fowler-Guggenheim adsorption model. The complex adsorption kinetics of streptavidin to biotinylated structures needs to be considered when these intermolecular bonds are employed in self-assembly and nanobiotechnology.     

Kinetics of ligand binding to membrane receptors from equilibrium fluctuation analysis of single binding events

Equilibrium fluctuation analysis of single binding events has been used to extract binding kinetics of ligand interactions with cell-membrane bound receptors. Time-dependent total internal reflection fluorescence (TIRF) imaging was used to extract residence-time statistics of fluorescently stained liposomes derived directly from cell membranes upon their binding to surface-immobilized antibody fragments. The dissociation rate constants for two pharmaceutical relevant antibodies directed against different B-cell expressed membrane proteins was clearly discriminated, and the affinity of the interaction could be determined by inhibiting the interaction with increasing concentrations of soluble antibodies. The single-molecule sensitivity made the analysis possible without overexpressed membrane proteins, which makes the assay attractive in early drug-screening applications.     

基于框架核酸的细胞表面受体配体相互作用研究进展

细胞表面受体与配体之间的特异性相互作用在细胞生物学过程中起着重要作用。然而,与均相溶液不同,受体分子在细胞膜上的分布是非连续的、动态的,因此细胞表面的受体配体相互作用通常呈现复杂的非线性结合模式。框架核酸作为一类具有确定几何形状的DNA纳米支架,可用于多价配体的偶联,为深入揭示受体配体相互作用机制提供了可靠的工具。利用框架核酸纳米分辨率的可寻址特性,可实现对配体数目、间距及空间构象等参数的精确调控,进而研究细胞表面受体配体的结合特性及影响因素,优化结合条件最终实现高效的分子识别及靶向治疗。本文综述了基于框架核酸的细胞表面受体配体相互作用研究进展,通过探讨细胞表面受体配体相互作用的重要影响因素及生物学应用,对该研究领域的发展前景和未来趋势予以展望。     

Activation of G-protein-coupled receptors in cell-derived plasma membranes supported on porous beads

G-protein-coupled receptors (GPCRs) are ubiquitous mediators of signal transduction across cell membranes and constitute a very important class of therapeutic targets. In order to study the complex biochemical signaling network coupling to the intracellular side of GPCRs, it is necessary to engineer and control the downstream signaling components, which is difficult to realize in living cells. We have developed a bioanalytical platform enabling the study of GPCRs in their native membrane transferred inside-out from live cells to lectin-coated beads, with both membrane sides of the receptor being accessible for molecular interactions. Using heterologously expressed adenosine A(2A) receptor carrying a yellow fluorescent protein, we showed that the tethered membranes comprised fully functional receptors in terms of ligand and G protein binding. The interactions between the different signaling partners during the formation and subsequent dissociation of the ternary signaling complex on single beads could be observed in real time using multicolor fluorescence microscopy. This approach of tethering inside-out native membranes accessible from both sides is straightforward and readily applied to other transmembrane proteins. It represents a generic platform suitable for ensemble as well as single-molecule measurements to investigate signaling processes at plasma membranes.     

Single-molecule force spectroscopy study of interaction between transforming growth factor beta1 and its receptor in living cells

Transforming growth factor beta1 (TGF-beta1) regulates many important cellular processes such as cell proliferation, differentiation, and apoptosis, etc. Its signaling is initiated by binding to and bringing together TGF-beta type II receptor (TbetaRII) and type I receptor (TbetaRI). However, it is not fully understood how the TGF-beta1 ligand-receptor interaction occurs in living cells and what is the molecular mechanism of the signaling complex TGF-beta1/TbetaRII/TbetaRI formation. In this study, we have investigated the interaction between TGF-beta1 and its receptors in living cells with single-molecule force spectroscopy for the first time. By positioning TGF-beta1-modified atomic force microscope (AFM) tips on the cells expressing fluorescent protein tagged TGF-beta receptors, the living-cell force measurement was realized with a combined fluorescence microscope and AFM. We found that coexpression of TbetaRI with TbetaRII enhanced the binding force of TGF-beta1 with its receptors, whereas the expressed TbetaRI itself exhibited no binding affinity to TGF-beta1. Moreover, the unbinding dynamics of TGF-beta1/TbetaRII and TGF-beta1/TbetaRI/TbetaRII were investigated with dynamic force spectroscopy under different AFM loading rates. The dissociation rate constants of TGF-beta1 with its receptors as well as other parameters characterizing their dissociation pathways were obtained. The results suggested a more stable binding of TGF-beta1 with the receptor after TbetaRI is recruited and the important contribution of TbetaRI to the signaling complex formation during TGF-beta1 signaling.     

A Novel Approach for the Evaluation of Positive Cooperative Guest Binding: Kinetic Consequences of Structural Tightening

Cooperativity is one of the most relevant features displayed by biomolecules. Thus, one of the challenges in the field of supramolecular chemistry is to understand the mechanisms underlying cooperative binding effects. Traditionally, cooperativity has been related to multivalent receptors, but Williams et al. have proposed a different interpretation based on the strengthening of noncovalent interactions within receptors upon binding. According to such an interpretation, positive cooperative binding operates through structural tightening. Hence, a quite counterintuitive kinetic behavior for positively cooperative bound complexes may be postulated: the more stable the complex, the slower it is formed. Such a hypothesis was tested in a synthetic system in which positive cooperative binding was previously confirmed by calorimetric experiments. Indeed, a linear correlation between the thermodynamics (ΔG°) and the kinetics (ΔG^≠) of guest binding confirmed the expected behavior. These distinctive kinetics provide solid evidence of positive cooperative guest binding, which is particularly useful bearing in mind that kinetic experiments are frequently and accurately carried out in both synthetic and biological systems.     

Surface behavior and peptide-lipid interactions of the cyclic neuropeptide melanin concentrating hormone

The kinetics and the thermodynamics of melanin concentrating hormone (MCH) adsorption, penetration, and mixing with membrane components are reported. MCH behaved as a surface active peptide, forming stable monolayers at a lipid-free air-water interface, with an equilibrium spreading pressure, a collapse pressure, and a minimal molecular area of 11 mN/m, 13 mN/m, and 140 A (2), respectively. Additional peptide interfacial stabilization was achieved in the presence of lipids, as evidenced by the expansion observed at pi > pi sp in monolayers containing premixtures of MCH with zwitterionic or charged lipids. The MCH-monolayer association and dissociation rate constants were 9.52 x 10 (-4) microM (-1) min (-1) and 8.83 x 10 (-4) min (-1), respectively. The binding of MCH to the dpPC-water interface had a K d = 930 nM at 10 mN/m. MCH penetration in lipid monolayers occurred even up to pi cutoff = 29-32 mN/m. The interaction stability, binding orientation, and miscibility of MCH in monolayers depended on the lipid type, the MCH molar fraction in the mixture, and the molecular packing of the monolayer. This predicted its heterogeneous distribution between different self-separated membrane domains. Our results demonstrated the ability of MCH to incorporate itself into biomembranes and supports the possibility that MCH affects the activity of mechanosensitive membrane proteins through mechanisms unrelated with binding to specific receptors.     

Novel functions and binding mechanisms of carbohydrate-binding proteins determined by force measurements

Cell surface carbohydrates are important targets for many cell surface receptors, and they mediate crucial biological processes ranging from pathogen infectivity to neutrophil adhesion to drug targeting. A central challenge is to identify relationships between lectin architecture and function that influence the adhesion strength, avidity, and kinetics of receptor-glycan bonds. This information is central both to understanding recognition mechanisms and to developing effective therapeutic agents for drug targeting or for preventing infection. Increasingly, force probes are used to assess structure activity relationships of both the glycan ligands and the receptors that bind them, as well as molecular mechanisms underlying binding and adhesion. This review describes recent advances in the use of different force measurement techniques to quantify receptor-glycan bond parameters, and to identify novel features of molecular mechanisms underlying recognition and adhesion. The examples discussed focus in particular on single bond rupture, surface force measurements, and micropipette manipulation. This review emphasizes the often-unique information obtained from studies of lectin interactions with carbohydrate ligands that complement more common structure determinations and solution binding studies.     

Cooperatively Enhanced Receptors for Biomimetic Molecular Recognition

The concept of preorganization suggests that organizing a receptor around its guest during binding is detrimental, because the cost of conformational change is assumed to be paid out of the binding energy. Although this concept has historically guided the synthesis of a great many synthetic hosts, in recent years, chemists have begun to synthesize receptors that resemble proteins in their cooperative conformational changes. Such changes could enhance the host–guest interactions, in particular if the binding of the guest triggers previously unengaged noncovalent interactions within the host. These hosts, referred to as cooperatively enhanced receptors, corroborate with their biological counterparts to support the approach of creating high‐affinity receptors through the combined strategies of cooperativity and preorganization. Solvents, often the invisible participants of any solution‐based supramolecular process, should be properly considered in the design of synthetic receptors, whether preorganized or cooperatively enhanced.     

Hormone receptor mobility and catecholamine binding in membranes. A theoretical model.

[3H]-Catecholamine binding to intact cells, isolated cell membranes, and to several isolated macromolecules has been shown by several laboratories to be neither stereospecific nor inhibited by known beta-antagonists. Since additional evidence indicates that this binding is not an artifact (i.e. due neither to the binding of a catecholamine oxidation product nor hormone binding to a catabolic enzyme such as COMT), the question remains as to whether this represents binding to a bona fide membrane receptor. Because all ligands which bind strongly or compete for this binding possess a catechol group, one possible explanation is that the binding affinity is primarily determined by the catechol moiety, whereas the correct stereoisomer of the side chain is necessary to activate the receptor. Thus, although binding is a necessary condition for hormone action, the necessary and sufficient condition for activation of adenyl cyclase is both the catechol group and the correct stereoisomer of the side chain. A theoretical model is developed here to provide a quantitative basis for this hypothesis. This model extends the current concept of distinct subunits in the adenyl cyclase system by separating the receptors from the catalytic sites and placing them at separate locations within the membrane. Utilizing the spare receptor model of Furchgott, and the mobility of macromolecules within a "lipid sea," the appropriate equations to predict both hormone binding and enzyme activation are derived. Using the observed affinity constants from catecholamine binding studies, it is then shown that this model can predict the experimental observation and hence explain the apparent dichotomy arising from binding enzyme activation studies.     

862 — The sequence and energetics of cell membrane transductive coupling to intracellular enzyme systems

We have shown that weak oscillating electromagnetic fields in the pericellular environment modulate key steps in coupling of signals from humoral stimuli at cell surface receptors to intracellular systems. This paper summarizes evidence that enzymatic activity within the cell provides sensitive molecular markers about both the sequence and the energetics of transmembrane coupling mechanisms. As research tools, these imposed fields appear to offer unique opportunities for understanding highly non-linear, non-equilibrium aspects of these coupling mechanisms, including the basis for amplification of weak pericellular stimuli to achieve an energetic threshold in signaling to intracellular enzyme systems.A three-stage model of membrane transductive coupling is proposed: a first stage in which weak pericellular electrochemical oscillations and binding of humoral stimulating molecules at receptor sites initiates a highly cooperative modification of calcium binding, a second stage involving transmission of signals initiated at receptor sites to the cell interior; and a third stage dealing with intracellular response to the transmembrane signal.Low frequency pulsed magnetic fields modulate stimulation of adenylate cyclase by parathyroid hormone (PTH) in bone cells. From collateral studies of field effects on PTH-binding to its receptor and from studies of fluoride activation of adenylate cyclase, there is evidence that an important field action is on membrane coupling proteins between receptors for PTH and adenylate cyclase.In addition to an action on adenylate cyclase, weak pericellular fields modulate activity of messenger enzymes, the protein kinases, and of an enzyme essential for cell growth, omithine decarboxylase. In human lymphocytes, cAMP-independent protein kinases are transiently inhibited by exposure to weak (athermal) microwave fields sinusoidally modulated at 16 Hz. In liver and ovary cells exposed to the same fields, and in bone cells exposed to low frequency pulsed magnetic fields, omithine decarboxylase activity is increased.Experimental data and models interrelating the pericellular electrochemical environment, cancer-promoting phorbol esters and activities of protein kinases and ornithine decarboxylase are discussed.     

A fractal analysis of analyte-estrogen receptor binding and dissociation kinetics using biosensors: environmental effects

A fractal analysis is used to model the binding and dissociation kinetics between analytes in solution and estrogen receptors (ER) immobilized on a sensor chip of a surface plasmon resonance (SPR) biosensor. Both cases are analyzed: unliganded as well as liganded. The influence of different ligands is also analyzed. A better understanding of the kinetics provides physical insights into the interactions and suggests means by which appropriate interactions (to promote correct signaling) and inappropriate interactions such as with xenoestrogens (to minimize inappropriate signaling and signaling deleterious to health) may be better controlled. The fractal approach is applied to analyte-ER interaction data available in the literature. Numerical values obtained for the binding and the dissociation rate coefficients are linked to the degree of roughness or heterogeneity (fractal dimension, D(f)) present on the biosensor chip surface. In general, the binding and the dissociation rate coefficients are very sensitive to the degree of heterogeneity on the surface. For example, the binding rate coefficient, k, exhibits a 4.60 order of dependence on the fractal dimension, D(f), for the binding of unliganded and liganded VDR mixed with GST-RXR in solution to Spp-1 VDRE (1,25-dihydroxyvitamin D(3) receptor element) DNA immobilized on a sensor chip surface (Cheskis and Freedman, Biochemistry 35 (1996) 3300-3318). A single-fractal analysis is adequate in some cases. In others (that exhibit complexities in the binding or the dissociation curves) a dual-fractal analysis is required to obtain a better fit. A predictive relationship is also presented for the ratio K(A)(=k/k(d)) as a function of the ratio of the fractal dimensions (D(f)/D(fd)). This has biomedical and environmental implications in that the dissociation and binding rate coefficients may be used to alleviate deleterious effects or enhance beneficial effects by selective modulation of the surface. The K(A) exhibits a 112-order dependence on the ratio of the fractal dimensions for the ligand effects on VDR-RXR interaction with specific DNA.     

A diverse set of oligomeric class II MHC-peptide complexes for probing T-cell receptor interactions

BACKGROUND: T-cells are activated by engagement of their clonotypic cell surface receptors with peptide complexes of major histocompatibility complex (MHC) proteins, in a poorly understood process that involves receptor clustering on the membrane surface. Few tools are available to study the molecular mechanisms responsible for initiation of activation processes in T-cells. RESULTS: A topologically diverse set of oligomers of the human MHC protein HLA-DR1, varying in size from dimers to tetramers, was produced by varying the location of an introduced cysteine residue and the number and spacing of sulfhydryl-reactive groups carried on novel and commercially available cross-linking reagents. Fluorescent probes incorporated into the cross-linking reagents facilitated measurement of oligomer binding to the T-cell surface. Oligomeric MHC-peptide complexes, including a variety of MHC dimers, trimers and tetramers, bound to T-cells and initiated T-cell activation processes in an antigen-specific manner. CONCLUSION: T-cell receptor dimerization on the cell surface is sufficient to initiate intracellular signaling processes, as a variety of MHC-peptide dimers differing in intramolecular spacing and orientation were each able to trigger early T-cell activation events. The relative binding affinities within a homologous series of MHC-peptide oligomers suggest that T-cell receptors may rearrange in the plane of the membrane concurrent with oligomer binding.     

A model for describing the thermodynamics of multivalent host-guest interactions at interfaces

A model has been described for interpreting the binding of multivalent molecules to interface-immobilized monovalent receptors through multiple, independent interactions. It is based on the concept of effective concentration, C(eff), which has been developed before for multivalent binding in solution and which incorporates effects of lengths and flexibilities of linkers between interacting sites. The model assumes: (i). the interactions are independent, (ii). the maximum number of interactions, p(max), is known, (iii). C(eff) is estimated from (simple) molecular models. Simulations of the thermodynamics and kinetics of multivalent host-guest binding to interfaces have been discussed, and competition with a monovalent competitor in solution has been incorporated as well. The model was successfully used to describe the binding of a divalent guest to self-assembled monolayers of a cyclodextrin host. The adsorption data of more complex guest-functionalized dendrimers, for which p(max) was not known beforehand, was interpreted as well. Finally, it has been shown that the model can aid to deconvolute contributions of multivalency and cooperativity to stability enhancements observed for the adsorption of multivalent molecules to interfaces.     

The Role of Lipids in Allosteric Modulation of Dopamine D2 Receptor—In Silico Study

The dopamine D₂ receptor, belonging to the class A G protein-coupled receptors (GPCRs), is an important drug target for several diseases, including schizophrenia and Parkinson’s disease. The D₂ receptor can be activated by the natural neurotransmitter dopamine or by synthetic ligands, which in both cases leads to the receptor coupling with a G protein. In addition to receptor modulation by orthosteric or allosteric ligands, it has been shown that lipids may affect the behaviour of membrane proteins. We constructed a model of a D₂ receptor with a long intracellular loop (ICL3) coupled with G_iα1 or G_iα2 proteins, embedded in a complex asymmetric membrane, and simulated it in complex with positive, negative or neutral allosteric ligands. In this study, we focused on the influence of ligand binding and G protein coupling on the membrane–receptor interactions. We show that there is a noticeable interplay between the cell membrane, G proteins, D₂ receptor and its modulators.