D1 and D2 dopamine receptor expression is regulated by direct interaction with the chaperone protein calnexin

As for all proteins, G protein-coupled receptors (GPCRs) undergo synthesis and maturation within the endoplasmic reticulum (ER). The mechanisms involved in the biogenesis and trafficking of GPCRs from the ER to the cell surface are poorly understood, but they may involve interactions with other proteins. We have now identified the ER chaperone protein calnexin as an interacting protein for both D(1) and D(2) dopamine receptors. These protein-protein interactions were confirmed using Western blot analysis and co-immunoprecipitation experiments. To determine the influence of calnexin on receptor expression, we conducted assays in HEK293T cells using a variety of calnexin-modifying conditions. Inhibition of glycosylation either through receptor mutations or treatments with glycosylation inhibitors partially blocks the interactions with calnexin with a resulting decrease in cell surface receptor expression. Confocal fluorescence microscopy reveals the accumulation of D(1)-green fluorescent protein and D(2)-yellow fluorescent protein receptors within internal stores following treatment with calnexin inhibitors. Overexpression of calnexin also results in a marked decrease in both D(1) and D(2) receptor expression. This is likely because of an increase in ER retention because confocal microscopy revealed intracellular clustering of dopamine receptors that were co-localized with an ER marker protein. Additionally, we show that calnexin interacts with the receptors via two distinct mechanisms, glycan-dependent and glycan-independent, which may underlie the multiple effects (ER retention and surface trafficking) of calnexin on receptor expression. Our data suggest that optimal receptor-calnexin interactions critically regulate D(1) and D(2) receptor trafficking and expression at the cell surface, a mechanism likely to be of importance for many GPCRs.     

Receptor properties of the hemagglutinin of influenza virus and its polypeptide fragments

The receptor properties of influenza virus A/Kiev/59/79 R (H1N1) and a number of its polypeptide fragments containing the aminoacids (from the 1st to 272d) of the heavy chain were studied. Two kinds of radioimmunoassay were used to test hemagglutinin or its polypeptide fragment interactions with cellular receptors. The studied polypeptides and hemagglutinin are shown to be capable of specific interactions with the receptors on the cell surface. The main linear fragment of hemagglutinin recognizing cellular receptors is localized within a polypeptide fragment including 1st-272d aminoacids of the heavy chain of hemagglutinin. The breaks of all the the S-S linkages including the ones linearly and spatially close to the receptor "pocket" of the bridge 95-135 do not affect significantly the receptor properties of the polypeptide.     

Cooperative properties of hormone receptors in cell membranes

The binding of many polypeptide hormones to cell surface receptors does not appear to follow the law of mass action. While steady–state binding data are consistent in many cases with either heterogeneous populations of binding sites or interactions of the type known as negative cooperativity, study of the kinetics of dissociation of the hormone receptor complex allows an unambiguous demonstration of cooperative interactions. Negative cooperativity, which seems to be wide-spread among hormone receptors, provides exquisite sensitivity of the cell at low hormone concentrations while buffering against acutely elevated hormone levels. The molecular mechanisms underlying the cooperativity are still largely unknown. Cooperativity may stem from a conformational transition in individual receptors or involve receptor aggregation in the fluid membrane (clustering) or more extensive membrane phenomena. Thus, new models of hormone action must be considered which integrate the progress in our knowledge of both the complex mechanisms regulating hormone binding to their surface receptors, and the dynamic properties of the cell membrane.     

Identification of CC chemokine receptor 7 residues important for receptor activation

The binding pocket of family A GPCRs that bind small biogenic amines is well characterized. In this study we identify residues on CC chemokine receptor 7 (CCR-7) that are involved in agonist-mediated receptor activation but not in high affinity ligand binding. The mutations also affect the ability of the ligands to induce chemotaxis. Two of the residues, Lys3.33(137) and Gln5.42(227), are consistent with the binding pocket described for biogenic amines, while Lys3.26(130) and Asn7.32(305), are found at, or close to, the cell surface. Our observations are in agreement with findings from other peptide and chemokine receptors, which indicate that receptors that bind larger ligands contain contact sites closer to the cell surface in addition to the conventional transmembrane binding pocket. These findings also support the theory that chemokine receptors require different sets of interactions for high affinity ligand binding and receptor activation.     

Analysis of ligand-receptor interactions in cells by atomic force microscopy

Atomic force microscopy (AFM) increasingly has been used to analyse "receptor" function, either by using purified proteins ("molecular recognition microscopy") or, more recently, in situ in living cells. The latter approach has been enabled by the use of a modified commercial AFM, linked to a confocal microscope, which has allowed adhesion forces between ligands and receptors in cells to be measured and mapped, and downstream cellular responses analysed. We review the application of AFM to cell biology and, in particular, to the study of ligand-receptor interactions and draw examples from our own work and that of others to show the utility of AFM, including for the exploration of cell surface functionalities. We also identify shortcomings of AFM in comparison to "standard" methods, such as receptor auto-radiography or immuno-detection, that are widely applied in cell biology and pharmacological analysis.     

Temperature-sensitive differential affinity of TRAIL for its receptors. DR5 is the highest affinity receptor

TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines which induces apoptotic cell death in a variety of tumor cell lines. It mediates its apoptotic effects through one of two receptors, DR4 and DR5, which are members of of the TNF receptor family, and whose cytoplasmic regions contain death domains. In addition, TRAIL also binds to 3 "decoy" receptors, DcR2, a receptor with a truncated death domain, DcR1, a glycosylphosphatidylinositol-anchored receptor, and OPG a secreted protein which is also known to bind to another member of the TNF family, RANKL. However, although apoptosis depends on the expression of one or both of the death domain containing receptors DR4 and/or DR5, resistance to TRAIL-induced apoptosis does not correlate with the expression of the "decoy" receptors. Previously, TRAIL has been described to bind to all its receptors with equivalent high affinities. In the present work, we show, by isothermal titration calorimetry and competitive enzyme-linked immunosorbent assay, that the rank order of affinities of TRAIL for the recombinant soluble forms of its receptors is strongly temperature dependent. Although DR4, DR5, DcR1, and OPG show similar affinities for TRAIL at 4 degrees C, their rank-ordered affinities are substantially different at 37 degrees C, with DR5 having the highest affinity (K(D) 相似文献   

Virus–Receptor Interactions: The Key to Cellular Invasion

Virus–receptor interactions play a key regulatory role in viral host range, tissue tropism, and viral pathogenesis. Viruses utilize elegant strategies to attach to one or multiple receptors, overcome the plasma membrane barrier, enter, and access the necessary host cell machinery. The viral attachment protein can be viewed as the “key” that unlocks host cells by interacting with the “lock”—the receptor—on the cell surface, and these lock-and-key interactions are critical for viruses to successfully invade host cells. Many common themes have emerged in virus–receptor utilization within and across virus families demonstrating that viruses often target particular classes of molecules in order to mediate these events. Common viral receptors include sialylated glycans, cell adhesion molecules such as immunoglobulin superfamily members and integrins, and phosphatidylserine receptors. The redundancy in receptor usage suggests that viruses target particular receptors or “common locks” to take advantage of their cellular function and also suggests evolutionary conservation. Due to the importance of initial virus interactions with host cells in viral pathogenesis and the redundancy in viral receptor usage, exploitation of these strategies would be an attractive target for new antiviral therapeutics.     

The Src family of tyrosine protein kinases in hemopoietic signal transduction.

The Src family of tyrosine protein kinases represent an expanding class of closely related intracellular enzymes that participate in the signal transduction pathways of a variety of surface receptors. One of the more surprising aspects of the information relating Src protein kinases to receptor signaling is the apparent diversity of receptor types with which the Src-related enzymes are reported to interact physically or functionally. Traditional biochemical and genetic approaches have yielded much information regarding the interactions between the Src tyrosine protein kinases and other cellular proteins in defined cell types, and emerging technologies, most notably homologous recombination in embryonal stem cells to achieve gene "knockouts," are providing new insights into the participation of the Src-related gene products in signal transduction and development.     

The concept of a changing receptor concentration: implications for the theory of drug action

Drugs are considered to produce their effects on biological tissues either by altering some physical property of cells or by interacting with specific cellular components, called receptors. Most drugs and endogenous neurotransmitters act on highly selective receptors located on the outer surface membrane of cells. These receptors were believed, until recently, to be stationary on the cell surface and to be present in unvarying numbers. Consequently, most early theorists modeled the drug-receptor interaction on the basis of stationary and static receptor molecules. The substantial advances in our understanding of drug action based on these models have partly justified this view. However, recent electron microscopic studies have revealed the presence of structures, including "coated" pits and vesicles, that appear to provide a mechanism by which cell surface receptors might be internalized in a process of endocytosis. The precise intracellular fate of these internalized receptors is unknown, but based on present understanding, it seems reasonable to believe that some are destroyed intracellularly whereas others are recycled to the cell surface. The importance of such processes to pharmacologic theory is a new awareness of a cellular pathway that is capable of internalizing drugs, receptors, or both. The implications of such a process to the theory of drug action extends to some unexplained drug phenomena such as down regulation, drug tolerance, tachyphyllaxis, and partial agonism. We present herein the theoretical framework for a model of drug action that incorporates the possibility of receptor internalization and subsequent degradation, recycling, or replacement.     

Autocrine ligand binding to cell receptors. Mathematical analysis of competition by solution "decoys".

Autocrine ligands have been demonstrated to regulate cell proliferation, cell adhesion, and cell migration in a number of different systems and are believed to be one of the underlying causes of malignant cell transformation. Binding of these ligands to their cellular receptors can be compromised by diffusive transport of ligand away from the secreting cell. Exogenous addition of antibodies or solution receptors capable of competing with cellular receptors for these autocrine ligands has been proposed as a means of inhibiting autocrine-stimulated cell behavioral responses. Such "decoys" complicate cellular binding by offering alternative binding targets, which may also be capable of aiding or abating transport of the ligand away from the cell surface. We present a mathematical model incorporating autocrine ligand production and the presence of competing cellular and solution receptors. We elucidate effects of key system parameters including ligand diffusion rate, binding rate constants, cell density, and secretion rate on the ability of solution receptors to inhibit cellular receptor binding. Both plated and suspension cell systems are considered. An approximate analytical expression relating the key parameters to the critical concentration of solution "decoys" required for inhibition is derived and compared to the numerical calculations. We find that in order to achieve essentially complete inhibition of surface receptor binding, the concentration of decoys may need to be as much as four to eight orders of magnitude greater than the equilibrium disociation constant for ligand binding to surface receptors.     

Large-scale co-aggregation of fluorescent lipid probes with cell surface proteins

Large scale aggregation of fluorescein-labeled immunoglobulin E (IgE) receptor complexes on the surface of RBL cells results in the co- aggregation of a large fraction of the lipophilic fluorescent probe 3,3'-dihexadecylindocarbocyanine (diI) that labels the plasma membranes much more uniformly in the absence of receptor aggregation. Most of the diI molecules that are localized in patches of aggregated receptors have lost their lateral mobility as determined by fluorescence photobleaching recovery. The diI outside of patches is mobile, and its mobility is similar to that in control cells without receptor aggregates. It is unlikely that the co-aggregation of diI with IgE receptors is due to specific interactions between these components, as two other lipophilic probes of different structures are also observed to redistribute with aggregated IgE receptors, and aggregation of two other cell surface antigens also results in the coredistribution of diI at the RBL cell surface. Quantitative analysis of CCD images of labeled cells reveals some differences in the spatial distributions of co- aggregated diI and IgE receptors. The results indicate that cross- linking of specific cell surface antigens causes a substantial change in the organization of the plasma membrane by redistributing pre- existing membrane domains or causing their formation.     

Dimerization of internalized epidermal growth factor receptors.

Binding of epidermal growth factor (EGF) to cell surface EGF receptors initiates the formation of the receptor homodimers that can be detected by covalent cross-linking in intact cells or in detergent-solubilized cell extracts. Low pH dissociation of EGF from surface receptors results in immediate monomerization of receptor dimers. Using chemical cross-linking during mild permeabilization or cell solubilization, we have detected dimers of internalized EGF receptors in human carcinoma A-431 cells and transfected NIH 3T3 cells that express human EGF receptors. The percentage of internalized cross-linked receptor dimers was similar to that observed for surface EGF receptors. Furthermore, at the time of maximal accumulation of EGF-receptor complexes within the endosomal compartment (10-15 min of incubation at 37 degrees C), both the dimeric and monomeric forms of the EGF receptor are tyrosine-phosphorylated to the same extent as surface dimer and monomer species. In transfected NIH 3T3 cells, the level of dimerized and internalized kinase-negative EGF receptors was not different from that observed for wild-type receptors. These data suggest that for some time after internalization EGF does not dissociate from its receptor and indicate that a receptor conformation is preserved intracellularly that allows maintenance of receptor-receptor interactions and tyrosine kinase activity.     

Self-association and raft localization of functional luteinizing hormone receptors

Membrane motions of LH receptors following binding of hormone agonists are consistent with hormone-driven aggregation. It is increasingly apparent that G protein-coupled receptors, including the LH receptor, are engaged in dynamic interactions with one another and other membrane components. These interactions are governed, in part, by a number of factors including whether the receptor has bound ligand, whether the receptor is capable of transducing a hormone-mediated signal, and the nature of the membrane environment within which the receptor is found. Microscopic methods, including laser-optical techniques, are ideally suited to probe dynamic events on cell membranes and provide an opportunity to examine interactions between receptors and other membrane components on viable cells. We and others have used a variety of techniques, some of which are summarized below, to examine functional and nonfunctional LH receptors on viable cells and the membrane environment of these receptors during cell signaling events.     

Adenovirus RIDbeta subunit contains a tyrosine residue that is critical for RID-mediated receptor internalization and inhibition of Fas- and TRAIL-induced apoptosis

The adenovirus-encoded receptor internalization and degradation (RID) protein (previously named E3-10.4K/14.5K), which is composed of RIDalpha and RIDbeta subunits, down-regulates a number of cell surface receptors in the tumor necrosis factor (TNF) receptor superfamily, namely Fas, TRAIL receptor 1, and TRAIL receptor 2. Down-regulation of these "death" receptors protects adenovirus-infected cells from apoptosis induced by the death receptor ligands Fas ligand and TRAIL. RID also down-regulates certain tyrosine kinase cell surface receptors, especially the epidermal growth factor receptor (EGFR). RID-mediated Fas and EGFR down-regulation occurs via endocytosis of the receptors into endosomes followed by transport to and degradation within lysosomes. However, the molecular interactions underlying this function of RID are unknown. To investigate the molecular determinants of RIDbeta that are involved in receptor down-regulation, mutations within the cytoplasmic tail of RIDbeta were constructed and the mutant proteins were analyzed for their capacity to internalize and degrade Fas and EGFR and to protect cells from death receptor ligand-induced apoptosis. The results demonstrated the critical nature of a tyrosine residue near the RIDbeta C terminus; mutation of this residue to alanine abolished RID function. Mutating the tyrosine to phenylalanine did not abolish the function of RID, arguing that phosphorylation of the tyrosine is not required for function. These data suggest that this tyrosine residue forms part of a tyrosine-based sorting signal (Yxxphi). Additional mutations that target another potential sorting motif and several possible protein-protein interaction motifs had no discernible effect on RID function. It was also demonstrated that mutation of serine 116 to alanine eliminated phosphorylation of RIDbeta but did not affect any of the functions of RID that were examined. These results suggest a model in which the tyrosine-based sorting signal in RID plays a role in RID's ability to down-regulate receptors.     

Transmembrane peptides as inhibitors of ErbB receptor signaling

Receptor tyrosine kinases have a single transmembrane (TM) segment that is usually assumed to play a passive role in ligand-induced dimerization and activation of the receptor. However, mutations within some of these receptors, and recent studies with the epidermal growth factor (EGF) and ErbB2 receptors have indicated that interactions between TM domains do contribute to stabilization of ligand-independent and/or ligand-induced receptor dimerization and activation. One consequence of the importance of these interactions is that short hydrophobic peptides corresponding to these domains should act as specific inhibitors. To test this hypothesis, we constructed expression vectors encoding short fusion peptides encompassing native or mutated TM domains of the EGF, ErbB2, and insulin receptors. In human cell lines overexpressing the wild-type EGF receptor or ErbB2, we observed that the peptides are expressed at the cell surface and that they inhibit specifically the autophosphorylation and signaling pathway of their cognate receptor. Identical results were obtained with peptides chemically synthesized. Mechanism of action involves inhibition of dimerization of the receptors as shown by the lack of effects of mutant nondimerizing sequences, completed by density centrifugation and covalent cross-linking experiments. Our findings stress the role of TM domain interactions in ErbB receptor function, and possibly for other single-spanning membrane proteins.     

A novel system for convenient detection of low-affinity receptor-ligand interactions: chelator-lipid liposomes engrafted with recombinant CD4 bind to cells expressing MHC class II

The interactions of cell surface receptors with their ligands, crucial for initiating many immunological responses, are often stabilized by receptor dimerization/oligomerization, and by multimeric interactions between receptors on one cell with their ligands or cognate receptors on the apposing cell. Current techniques for studying receptor-ligand interactions, however, do not always allow receptors to move laterally to enable dimerization/ oligomerization, or to interact multimerically with ligands on cell surfaces. For these reasons detection of low- affinity receptor-ligand interactions has been difficult. Utilizing a novel chelator-lipid, nitrilotriacetic acid di-tetradecylamine (NTA-DTDA), we have developed a convenient liposome system for directly detecting low-affinity receptor-ligand interactions. Our studies using recombinant soluble forms of murine CD40 and B7.1, and murine and human CD4, each possessing a hexhistidine tag, showed that these proteins can be anchored or 'engrafted' directly onto fluorescently labelled liposomes via a metal-chelating linkage with NTA-DTDA, permitting them to undergo dimerization/oligomerization and multimeric binding with ligands on cells. Fluorescence- activated cell sorter (FACS) analyses demonstrated that while there is little if any binding of soluble forms of murine CD40 and B7.1, and murine and human CD4 to cells, engrafted liposomes bind specifically to cells expressing the appropriate cognate receptor, often giving a fluorescence 4-6-fold above control cells. Such liposomes could detect directly the low-affinity interaction of murine CD40 and B7.1 with CD154- and CD28-expressing cells, respectively, and the interaction of CD4 with MHC Class II, which has hitherto defied direct detection except through mutational analysis and mAb blocking studies.     

Kinetic versus allosteric mechanisms to explain insurmountable antagonism and delayed ligand dissociation

The present review addresses the theories which have been advanced to explain experimental observations dealing with insurmountable antagonism and accelerated radioligand dissociation in the presence of an excess unlabelled ligand. We came to the perception that, for each of these phenomena, the theories can be placed into two distinctive categories. The "kinetic" interpretations attribute these phenomena to, respectively, the ability of antagonists to form long-lasting complexes with their cognate receptor and the ability of dissociated ligands to bind again to the same or neighbouring receptors rather than to diffuse away from the cell surface. On the other hand, these observations can also be explained by negative allosteric interactions among topographically distinct ligand binding sites at the same receptor or di/multimeric receptor complex.     

Specific capture of mammalian cells by cell surface receptor binding to ligand immobilized on gold thin films

Aldehyde-terminated self-assembled monolayers (SAMs) on gold surfaces were modified with proteins and employed to capture intact living cells through specific ligand-cell surface receptor interactions. In our model system, the basic fibroblast growth factor (bFGF) binding receptor was targeted on baby hamster kidney (BHK-21) cells. Negative control and target proteins were immobilized on a gold surface by coupling protein primary amines to surface aldehyde groups. Cell-binding was monitored by phase contrast microscopy or surface plasmon resonance (SPR) imaging. The specificity of the receptor-ligand interaction was confirmed by the lack of cell binding to the negative control proteins, cytochrome c and insulin, and by the disruption of cell binding by treatment with heparitinase to destroy heparan sulfate which plays an essential role in the binding of bFGF to FGF receptors. This approach can simultaneously probe a large number of receptor-ligand interactions in cell populations and has potential for targeting and isolating cells from mixtures according to the receptors expressed on their surface.