Physical responses of bacterial chemoreceptors

Chemoreceptors of the bacterium Escherichia coli are thought to form trimers of homodimers that undergo conformational changes upon ligand binding and thereby signal a cytoplasmic kinase. We monitored the physical responses of trimers in living cells lacking other chemotaxis proteins by fluorescently tagging receptors and measuring changes in fluorescence anisotropy. These changes were traced to changes in energy transfer between fluorophores on different dimers of a trimer: attractants move these fluorophores farther apart, and repellents move them closer together. These measurements allowed us to define the responses of bare receptor oligomers to ligand binding and compare them to the corresponding response in kinase activity. Receptor responses could be fit by a simple "two-state" model in which receptor dimers are in either active or inactive conformations, from which energy bias and dissociation constants could be estimated. Comparison with responses in kinase-activity indicated that higher-order interactions are dominant in receptor clusters.     

Adaptational modification and ligand occupancy have opposite effects on positioning of the transmembrane signalling helix of a chemoreceptor

Sensory systems adapt to persistent stimulation. In the transmembrane receptors of bacterial chemotaxis, adaptation is mediated by methylation at specific glutamyl residues in the cytoplasmic domain. Methylation counteracts effects of ligand binding on functional activities of that domain. Both ligand binding and adaptational modification are thought to act through conformational changes. As characterized for Escherichia coli chemoreceptors, a mechanistically crucial feature of the ligand-induced conformational change is piston sliding towards the cytoplasm of a signalling helix in the periplasmic/transmembrane domain. Adaptational modification could counteract this signalling movement by blocking its influence on the cytoplasmic domain or by reversing it. To investigate, we characterized effects of adaptational modification on the position of the signalling helix in chemoreceptor Trg using rates of disulphide formation between introduced cysteines. We utilized an intact cell procedure in which receptors were in their native, functional state. In vivo rates of disulphide formation between diagnostic cysteine pairs spanning a signalling helix interface changed as a function of adaptational modification. Strikingly, those changes were opposite those caused by ligand occupancy for each diagnostic pair tested. This suggests that adaptational modification resets the receptor complex to its null state by reversal of the conformational change generated by ligand binding.     

Stoichiometry of interaction between interferon gamma and its receptor.

The biological response of interferon gamma is mediated by binding to a specific cell-surface receptor. We investigated the stoichiometry of this binding using soluble receptors produced in prokaryotic and eukaryotic expression systems comprising the extracellular ligand-binding domain of the native protein. The ligand-receptor complexes were analyzed by cross-linking, chromatography, analytical ultracentrifugation and laser-light scattering. Cross-linking and chromatography showed that the stoichiometry of the interaction between ligand and receptor depends on the molar ratios of the two components mixed. All approaches confirmed that mixtures of ligand-receptor complexes are formed with one interferon-gamma dimer bound by one or two receptors. The soluble receptor produced in Escherichia coli mainly showed a ligand/receptor stoichiometry of 1:1, while the receptors produced in eukaryotic cells showed a stoichiometry of binding of 1:2. This apparent discrepancy is most likely due to the conformational heterogeneity of the Escherichia-coli-derived protein.     

Chemoreceptors in signalling complexes: shifted conformation and asymmetric coupling

Bacterial chemotaxis is mediated by signalling complexes of chemoreceptors, histidine kinase CheA and coupling protein CheW. Interactions in complexes profoundly affect the kinase. We investigated effects of these interactions on chemoreceptors by comparing receptors alone and in complexes. Assays of initial rates of methylation indicated that signalling complexes shifted receptor conformation towards the methylation-on, higher-ligand-affinity, kinase-off state, tuning receptors for greater sensitivity. In contrast, transmembrane and conformational signalling within chemoreceptors was essentially unaltered, consistent with other evidence identifying receptor dimers as the fundamental units of such signalling. In signalling complexes, coupling of ligand binding to kinase activity is cooperative and the dynamic range of kinase control expanded > 100-fold by receptor adaptational modification. We observed no cooperativity in influence of ligand on receptor conformation, only on kinase activity. However, receptor modification generated increased dynamic range in a stepwise fashion, partly in coupling ligand to receptor conformation and partly in coupling receptor conformation to kinase activity. Thus, receptors and kinase were not equivalently affected by interactions in signalling complexes or by ligand binding and adaptational modification, indicating asymmetrical coupling between them. This has implications for mechanisms of precise adaptation. Coupling might vary, providing a previously unappreciated locus for sensory control.     

Bioluminescence resonance energy transfer reveals ligand-induced conformational changes in CXCR4 homo- and heterodimers

Homo- and heterodimerization have emerged as prominent features of G-protein-coupled receptors with possible impact on the regulation of their activity. Using a sensitive bioluminescence resonance energy transfer system, we investigated the formation of CXCR4 and CCR2 chemokine receptor dimers. We found that both receptors exist as constitutive homo- and heterodimers and that ligands induce conformational changes within the pre-formed dimers without promoting receptor dimer formation or disassembly. Ligands with different intrinsic efficacies yielded distinct bioluminescence resonance energy transfer modulations, indicating the stabilization of distinct receptor conformations. We also found that peptides derived from the transmembrane domains of CXCR4 inhibited activation of this receptor by blocking the ligand-induced conformational transitions of the dimer. Taken together, our data support a model in which chemokine receptor homo- and heterodimers form spontaneously and respond to ligand binding as units that undergo conformational changes involving both protomers even when only one of the two ligand binding sites is occupied.     

Functional roles for the cytoplasmic domain of the type III transforming growth factor beta receptor in regulating transforming growth factor beta signaling

Transforming growth factor beta (TGF-beta) signals through three high affinity cell surface receptors, TGF-beta type I, type II, and type III receptors. The type III receptor, also known as betaglycan, binds to the type II receptor and is thought to act solely by "presenting" the TGF-beta ligand to the type II receptor. The short cytoplasmic domain of the type III receptor is thought to have no role in TGF-beta signaling because deletion of this domain has no effect on association with the type II receptor, or with the presentation role of the type III receptor. Here we demonstrate that the cytoplasmic domains of the type III and type II receptors interact specifically in a manner dependent on the kinase activity of the type II receptor and the ability of the type II receptor to autophosphorylate. This interaction results in the phosphorylation of the cytoplasmic domain of the type III receptor by the type II receptor. The type III receptor with the cytoplasmic domain deleted is able to bind TGF-beta, to bind the type II receptor, and to enhance TGF-beta binding to the type II receptor but is unable to enhance TGF-beta2 signaling, determining that the cytoplasmic domain is essential for some functions of the type III receptor. The type III receptor functions by selectively binding the autophosphorylated type II receptor via its cytoplasmic domain, thus promoting the preferential formation of a complex between the autophosphorylated type II receptor and the type I receptor and then dissociating from this active signaling complex. These studies, for the first time, elucidate important functional roles of the cytoplasmic domain of the type III receptor and demonstrate that these roles are essential for regulating TGF-beta signaling.     

Conformational change of the transmembrane helices II and IV of metabotropic glutamate receptor involved in G protein activation

G protein-coupled receptors are classified into several families on the basis of their amino acid sequences and the members of the same family exhibit sequence similarity but those of different families do not. In family 1 GPCRs such as rhodopsin and adrenergic receptor, extensive studies have revealed the stimulus-dependent conformational change of the receptor: the rearrangement of transmembrane helices III and VI is essential for G protein activation. In contrast, in family 3 GPCRs such as metabotropic glutamate receptor (mGluR), the inter-protomer relocation upon ligand binding has been observed but there is much less information about the structural changes of the transmsmbrane helices and the cytoplasmic domains. Here we identified constitutively active mutation sites at the cytoplasmic borders of helices II and IV of mGluR8 and successfully inhibited the G protein activation ability by engineering disulfide cross-linking between these cytoplasmic regions. The analysis of all possible single substitution mutants of these residues revealed that some steric interactions around these sites would be important to keep the receptor protein inactive. These results provided the model that the conformational changes at the cytoplasmic ends of helices II and IV of mGluR are involved in the efficient G protein coupling.     

Determination of an antibody-antigen binding constant by enzyme immunoassay and a theory for analysis of competitive binding of two ligands to heterogeneous receptor

A method for determining antigen-antibody binding constants by using enzyme-labeled antigens has been developed. In the measurement, enzyme-labeled and unlabeled antigens (Ag* and Ag) were allowed to compete in binding to the antibody (Ab) under conditions where Ag* much less than Ab much less than Ag. The data were analyzed according to a new theory developed for the analysis of competitive binding of two ligands to a heterogeneous receptor. The theory indicates that the binding degree of a labeled ligand measured at various concentrations of the receptor can be used to prepare a standard curve relating the binding degree of the labeled ligand and the average of the concentrations of the free receptor components which are in binding equilibrium with another unlabeled ligand. For homogeneous receptors, the method gives usual binding constants for the unlabeled ligand, but for heterogeneous receptors, it gives a new type of average binding constant for the unlabeled ligand in which the contribution of each receptor component is amplified in proportion to its affinity against the labeled ligand. This average binding constant was named the "affinity-average binding constant." A rabbit anti-blasticidin S (BLS) antiserum analyzed by the present method using beta-galactosidase-labeled BLS as the labeled ligand was found to be fairly homogeneous with respect to the affinity and to have a binding constant of 1.48 +/- 0.24 (S.D.) X 10(8) M-1 for unlabeled BLS.     

Ligand-induced rearrangement of the dimeric metabotropic glutamate receptor 1alpha

The extracellular domain of the metabotropic glutamate receptor 1alpha (mGluR1alpha) forms a dimer and the ligand, glutamate, induces a structural rearrangement in this domain. However, the conformational change in the cytoplasmic domain, which is critical for mGluR1alpha's interaction with G proteins, remains unclear. Here we investigated the ligand-induced conformational changes in the cytoplasmic domain by fluorescence resonance energy transfer (FRET) analysis of mGluR1alpha labeled with fluorescent protein(s) under total internal reflection field microscopy. Upon ligand binding, the intersubunit FRET efficiency between the second loops increased, whereas that between first loops decreased. In contrast, the intrasubunit FRET did not change clearly. These results show that ligand binding does not change the structure of each subunit, but does change the dimeric allocation of the cytoplasmic regions, which may underlie downstream signaling.     

Ligand selectivity and affinity of chemokine receptor CXCR1. Role of N-terminal domain

Glu-Leu-Arg ("ELR") CXC chemokines interleukin-8 (IL-8) and melanoma growth stimulatory activity (MGSA) recruit neutrophils by binding and activating two receptors, CXCR1 and CXCR2. CXCR1 is specific, binding only IL-8 with nanomolar affinity, whereas CXCR2 is promiscuous, binding all ELRCXC chemokines with high affinity. Receptor signaling consists of two events: interactions between the ligand N-terminal loop (N-loop) and receptor N-terminal domain (N-domain) residues (site I), and between the ligand N-terminal ELR and the receptor juxtamembrane domain (J-domain) residues (site II). It is not known how these interactions mediate ligand affinity and selectivity, and whether binding at one site influences binding and function at the other. Sequence analysis and structure-function studies have suggested that the receptor N-domain plays an important role in ligand selectivity. Here, we report ligand-binding properties and structural characteristics of the CXCR1 N-domain in solution and in detergent micelles that mimic the native membrane environment. We find that IL-8 binds the N-domain with significantly higher affinity in micelles than in solution (approximately 1 microM versus approximately 20 microM) and that MGSA does not bind the N-domain in solution but does in micelles with appreciable affinity (approximately 3 microM). We find that the N-domain is structured in micelles and that the entire N-domain interacts with the micelle in an extended fashion. We conclude that the micellar environment constrains the N-domain, and this conformational restraint influences its ligand-binding properties. Most importantly, our data suggest that for both ligands, site I interaction provides similar affinity and that differential coupling between site I and II interactions is responsible for the observed differences in affinity.     

Activation and desensitization induce distinct conformational changes at the extracellular-transmembrane domain interface of the glycine receptor

Most ligand-gated channels exhibit desensitization, which is the progressive fading of ionic current in the prolonged presence of agonist. This process involves conformational changes that close the channel despite continued agonist binding. Despite the physiological and pathological importance of desensitization, little is known about the conformational changes that underlie this process in any Cys-loop ion channel receptor. Here we employed voltage clamp fluorometry to identify conformational changes that occur with a similar time course as the current desensitization rate in both slow- and fast-desensitizing α1 glycine receptor chloride channels. Voltage clamp fluorometry provides a direct indication of conformational changes that occur in the immediate vicinity of residues labeled with environmentally sensitive fluorophores. We compared the rates of current desensitization and fluorescence changes at nine labeled extracellular sites in both wild type slow-desensitizing and mutated (A248L) fast-desensitizing glycine receptors. As labels attached to three sites at the interface between the ligand binding domain and transmembrane domain reported fluorescence responses that changed in parallel with the current desensitization rate, we concluded that they experienced local conformational changes associated with desensitization. These labeled sites included A52C in loop 2, Q219C in the pre-M1 domain, and M227C in the M1 domain. Activation and desensitization were accompanied by physically distinct conformational changes at each labeled site. Because activation is mediated by a specific reorganization of molecular interactions at the extracellular-transmembrane domain interface, we propose that desensitization is mediated by a distinct set of conformational changes that prevents this reorganization from occurring, thereby favoring channel closure.     

Comparative fluorescence resonance energy transfer analysis of metabotropic glutamate receptors: implications about the dimeric arrangement and rearrangement upon ligand bindings

Dimerization of G protein-coupled receptors has received much attention as a regulatory system of physiological function. Metabotropic glutamate receptors (mGluRs) are suitable models for studying the physiological significance of G protein-coupled receptor dimers because they form constitutive homodimers and function through dimeric rearrangement of their extracellular ligand binding domains. However, the molecular architecture of the transmembrane domains (TMDs) and their rearrangement upon agonist binding are still largely unknown. Here we show that the two helix Vs are arranged as the closest part in the dimeric TMDs and change their positions through synergistic control by the binding of two glutamates. The possibility that helix V is involved in an inter-protomer communication was first suggested by the finding that constitutively active mutation sites were identified on both sides of helix V. Then, comprehensive fluorescence resonance energy transfer (FRET) analysis using mGluRs whose cytoplasmic loops were labeled with donor and acceptor fluorescent proteins revealed that the third intracellular loop connecting helices V and VI of one protomer was in close proximity to the second and third intracellular loops of the other protomer and that all the intracellular loops became closer during the activation. Furthermore, FRET analysis of heterodimers in which only one protomer had ligand binding ability revealed the synergistic effect of the binding of two glutamates on the dimeric rearrangements of the TMD. Thus, the glutamate-dependent synergistic relocation of the helix Vs in the dimer is important for the signal flow from the extracellular ligand binding domain to the cytoplasmic surface of the mGluR.     

Cytohesins are cytoplasmic ErbB receptor activators

Signaling by ErbB receptors requires the activation of their cytoplasmic kinase domains, which is initiated by ligand binding to the receptor ectodomains. Cytoplasmic factors contributing to the activation are unknown. Here we identify members of the cytohesin protein family as such factors. Cytohesin inhibition decreased ErbB receptor autophosphorylation and signaling, whereas cytohesin overexpression stimulated receptor activation. Monitoring epidermal growth factor receptor (EGFR) conformation by anisotropy microscopy together with cell-free reconstitution of cytohesin-dependent receptor autophosphorylation indicate that cytohesins facilitate conformational rearrangements in the intracellular domains of dimerized receptors. Consistent with cytohesins playing a prominent role in ErbB receptor signaling, we found that cytohesin overexpression correlated with EGF signaling pathway activation in human lung adenocarcinomas. Chemical inhibition of cytohesins resulted in reduced proliferation of EGFR-dependent lung cancer cells in?vitro and in?vivo. Our results establish cytohesins as cytoplasmic conformational activators of ErbB receptors that are of pathophysiological relevance.     

The types II and III transforming growth factor-beta receptors form homo-oligomers

Affinity-labeling experiments have detected hetero-oligomers of the types I, II, and III transforming growth factor beta (TGF-beta) receptors which mediate intracellular signaling by TGF-beta, but the oligomeric state of the individual receptor types remains unknown. Here we use two types of experiments to show that a major portion of the receptor types II and III forms homo-oligomers both in the absence and presence of TGF-beta. Both experiments used COS-7 cells co-transfected with combinations of these receptors carrying different epitope tags at their extracellular termini. In immunoprecipitation experiments, radiolabeled TGF-beta was bound and cross-linked to cells co-expressing two differently tagged type II receptors. Sequential immunoprecipitations using anti-epitope monoclonal antibodies showed that type II TGF-beta receptors form homo-oligomers. In cells co- expressing epitope-tagged types II and III receptors, a low level of co- precipitation of the ligand-labeled receptors was observed, indicating that some hetero-oligomers of the types II and III receptors exist in the presence of ligand. Antibody-mediated cross-linking studies based on double-labeling immunofluorescence explored co-patching of the receptors at the cell surface on live cells. In cells co-expressing two differently tagged type II receptors or two differently tagged type III receptors, forcing one receptor into micropatches by IgG induced co- patching of the receptor carrying the other tag, labeled by noncross- linking monovalent Fab'. These studies showed that homo-oligomers of the types II and III receptors exist on the cell surface in the absence or presence of TGF-beta 1 or -beta 2. In cells co-expressing types II and III receptors, the amount of heterocomplexes at the cell surface was too low to be detected in the immunofluorescence co-patching experiments, confirming that hetero-oligomers of the types II and III receptors are minor and probably transient species.     

Differential hydrogen/deuterium exchange mass spectrometry analysis of protein-ligand interactions

Functional regulation of ligand-activated receptors is driven by alterations in the conformational dynamics of the protein upon ligand binding. Differential hydrogen/deuterium exchange (HDX) coupled with mass spectrometry has emerged as a rapid and sensitive approach for characterization of perturbations in conformational dynamics of proteins following ligand binding. While this technique is sensitive to detecting ligand interactions and alterations in receptor dynamics, it also can provide important mechanistic insights into ligand regulation. For example, HDX has been used to determine a novel mechanism of ligand activation of the nuclear receptor peroxisome proliferator activated receptor-γ, perform detailed analyses of binding modes of ligands within the ligand-binding pocket of two estrogen receptor isoforms, providing insight into selectivity, and helped classify different types of estrogen receptor-α ligands by correlating their pharmacology with the way they interact with the receptor based solely on hierarchical clustering of receptor HDX signatures. Beyond small-molecule-receptor interactions, this technique has also been applied to study protein-protein complexes, such as mapping antibody-antigen interactions. In this article, we summarize the current state of the differential HDX approaches and the future outlook. We summarize how HDX analysis of protein-ligand interactions has had an impact on biology and drug discovery.     

Cytokine receptors and signal transduction.

Most of the recently cloned cytokine receptors that operate in the immune and hematopoietic systems contain no tyrosine kinase domains in their cytoplasmic regions, unlike the family of growth factor receptors defined earlier. However, they can be assigned to several new types of receptor families based on structural similarities among them. It is characteristic of these receptors that many of them require a receptor-associated molecule in order to achieve high-affinity ligand binding and/or transmission of cytoplasmic signals. Receptor-associated molecules have been found that transduce cytoplasmic signals and are shared by different cytokine receptors. Phosphorylation of the receptors and of various cytoplasmic proteins after ligand stimulation seems to be a common event in cytokine systems. Insight into the pleiotropic and redundant nature of cytokine action is provided by the discovery of several new cytokine receptor families and of shared signal transduction molecules and by the idea that several cytoplasmic kinases may be able to functionally substitute for one another in transmitting cytokine signals.     

Plasmon-waveguide resonance spectroscopy: a new tool for investigating signal transduction by G-protein coupled receptors

Plasmon-waveguide resonance (PWR) spectroscopy provides a highly sensitive method for characterizing the kinetics, affinities and conformational changes involved in ligand binding to G-protein coupled receptors, without the need for radioactive or other labeling strategies. In the case of the cloned delta-opioid receptor from human brain incorporated into a lipid bilayer, we have shown that affinities determined in this way are consistent with those measured by standard binding procedures using membranes or whole cells containing the receptors, and that the spectral and kinetic properties of the binding processes allow facile distinction between agonist, inverse agonist, and antagonist ligands. We have also shown by direct measurements that G-protein binding affinities and the ability to undergo GTP/GDP exchange are dependent upon the type of ligand pre-bound to the receptor. PWR spectroscopy thus provides a powerful new approach to investigating signal transduction in biological membrane systems.     

Signalling‐dependent interactions between the kinase‐coupling protein CheW and chemoreceptors in living cells

Chemical signals sensed on the periplasmic side of bacterial cells by transmembrane chemoreceptors are transmitted to the flagellar motors via the histidine kinase CheA, which controls the phosphorylation level of the effector protein CheY. Chemoreceptor arrays comprise remarkably stable supramolecular structures in which thousands of chemoreceptors are networked through interactions between their cytoplasmic tips, CheA, and the small coupling protein CheW. To explore the conformational changes that occur within this protein assembly during signalling, we used in vivo cross‐linking methods to detect close interactions between the coupling protein CheW and the serine receptor Tsr in intact Escherichia coli cells. We identified two signal‐sensitive contacts between CheW and the cytoplasmic tip of Tsr. Our results suggest that ligand binding triggers changes in the receptor that alter its signalling contacts with CheW (and/or CheA).     

Subtypes of the somatostatin receptor assemble as functional homo- and heterodimers

The existence of receptor dimers has been proposed for several G protein-coupled receptors. However, the question of whether G protein-coupled receptor dimers are necessary for activating or modulating normal receptor function is unclear. We address this question with somatostatin receptors (SSTRs) of which there are five distinct subtypes. By using transfected mutant and wild type receptors, as well as endogenous receptors, we provide pharmacological, biochemical, and physical evidence, based on fluorescence resonance energy transfer analysis, that activation by ligand induces SSTR dimerization, both homo- and heterodimerization with other members of the SSTR family, and that dimerization alters the functional properties of the receptor such as ligand binding affinity and agonist-induced receptor internalization and up-regulation. Double label confocal fluorescence microscopy showed that when SSTR1 and SSTR5 subtypes were coexpressed in Chinese hamster ovary-K1 cells and treated with agonist they underwent internalization and were colocalized in cytoplasmic vesicles. SSTR5 formed heterodimers with SSTR1 but not with SSTR4 suggesting that heterodimerization is a specific process that is restricted to some but not all receptor subtype combinations. Direct protein interaction between different members of the SSTR subfamily defines a new level of molecular cross-talk between subtypes of the SSTR and possibly related receptor families.