A Positive Cooperativity Binding Model between Ly49 Natural Killer Cell Receptors and the Viral Immunoevasin m157: KINETIC AND THERMODYNAMIC STUDIES*

Natural killer (NK) cells discriminate between healthy and virally infected or transformed cells using diverse surface receptors that are both activating and inhibitory. Among them, the homodimeric Ly49 NK receptors, which can adopt two distinct conformations (backfolded and extended), are of particular importance for detecting cells infected with mouse cytomegalovirus (CMV) via recognition of the viral immunoevasin m157. The interaction of m157 with activating (Ly49H) and inhibitory (Ly49I) receptors governs the spread of mouse CMV. We carried out kinetic and thermodynamic experiments to elucidate the Ly49/m157 binding mechanism. Combining surface plasmon resonance, fluorescence anisotropy, and circular dichroism (CD), we determined that the best model to describe both the Ly49H/m157 and Ly49I/m157 interactions is a conformational selection mechanism where only the extended conformation of Ly49 (Ly49*) is able to bind the first m157 ligand followed by binding of the Ly49*/m157 complex to the second m157. The interaction is characterized by strong positive cooperativity such that the second m157 binds the Ly49 homodimer with a 1000-fold higher sequential constant than the first m157 (∼10⁸ versus ∼10⁵ m⁻¹). Using far-UV CD, we obtained evidence for a conformational change in Ly49 upon binding m157 that could explain the positive cooperativity. The rate-limiting step of the overall mechanism is a conformational transition in Ly49 from its backfolded to extended form. The global thermodynamic parameters from the initial state (backfolded Ly49 and m157) to the final state (Ly49*/(m157)₂) are characterized by an unfavorable enthalpy that is compensated by a favorable entropy, making the interaction spontaneous.     

Probing Natural Killer Cell Education by Ly49 Receptor Expression Analysis and Computational Modelling in Single MHC Class I Mice

Murine natural killer (NK) cells express inhibitory Ly49 receptors for MHC class I molecules, which allows for “missing self” recognition of cells that downregulate MHC class I expression. During murine NK cell development, host MHC class I molecules impose an “educating impact” on the NK cell pool. As a result, mice with different MHC class I expression display different frequency distributions of Ly49 receptor combinations on NK cells. Two models have been put forward to explain this impact. The two-step selection model proposes a stochastic Ly49 receptor expression followed by selection for NK cells expressing appropriate receptor combinations. The sequential model, on the other hand, proposes that each NK cell sequentially expresses Ly49 receptors until an interaction of sufficient magnitude with self-class I MHC is reached for the NK cell to mature. With the aim to clarify which one of these models is most likely to reflect the actual biological process, we simulated the two educational schemes by mathematical modelling, and fitted the results to Ly49 expression patterns, which were analyzed in mice expressing single MHC class I molecules. Our results favour the two-step selection model over the sequential model. Furthermore, the MHC class I environment favoured maturation of NK cells expressing one or a few self receptors, suggesting a possible step of positive selection in NK cell education. Based on the predicted Ly49 binding preferences revealed by the model, we also propose, that Ly49 receptors are more promiscuous than previously thought in their interactions with MHC class I molecules, which was supported by functional studies of NK cell subsets expressing individual Ly49 receptors.     

Functional consequences of natural sequence variation of murine cytomegalovirus m157 for Ly49 receptor specificity and NK cell activation

The Ly49H activating receptor on C57BL/6 (B6) NK cells plays a key role in early resistance to murine cytomegalovirus (MCMV) infection through specific recognition of the MCMV-encoded MHC class I-like molecule m157 expressed on infected cells. The m157 molecule is also recognized by the Ly49I inhibitory receptor from the 129/J mouse strain. The m157 gene is highly sequence variable among MCMV isolates, with many m157 variants unable to bind Ly49H(B6). In this study, we have sought to define if m157 variability leads to a wider spectrum of interactions with other Ly49 molecules and if this modifies host susceptibility to MCMV. We have identified novel m157-Ly49 receptor interactions, involving Ly49C inhibitory receptors from B6, BALB/c, and NZB mice, as well as the Ly49H(NZB) activation receptor. Using an MCMV recombinant virus in which m157(K181) was replaced with m157(G1F), which interacts with both Ly49H(B6) and Ly49C(B6), we show that the m157(G1F)-Ly49C interactions cause no apparent attenuating effect on viral clearance in B6 mice. Hence, when m157 can bind both inhibitory and activation NK cell receptors, the outcome is still activation. Thus, these data indicate that whereas m157 variants predominately interact with inhibitory Ly49 receptors, these interactions do not profoundly interfere with early NK cell responses.     

Critical residues at the Ly49 natural killer receptor's homodimer interface determine functional recognition of m157, a mouse cytomegalovirus MHC class I-like protein

NK cell function is regulated by Ly49 receptors in mice and killer cell Ig-like receptors in humans. Although inhibitory Ly49 and killer cell Ig-like receptors predominantly ligate classical MHC class I molecules, recent studies suggest that their activating counterparts recognize infection. The quintessential example is resistance to the mouse CMV in C57BL/6 mice, which depends on the functional recognition of m157, a mouse CMV-encoded MHC class I-like molecule, by Ly49H, an activating NK cell receptor. We have taken advantage of the natural variation in closely related members of the Ly49C-like receptors and the availability of Ly49 crystal structures to understand the molecular determinants of the Ly49H-m157 interaction and to identify amino acid residues discriminating between m157 binding and nonbinding receptors. Using a site-directed mutagenesis approach, we have targeted residues conserved in receptors binding to m157 (Ly49H and Ly49I(129)) but different from receptors lacking m157 recognition (Ly49C, Ly49I(B6), and Ly49U). Wild-type and mutant receptors were transfected into reporter cells, and physical binding as well as functional activation by m157 was studied. Our findings suggested that the Ly49 MHC class I contact site 2, I226, may not be involved in m157 binding. In contrast, residue Y146 and G151, mapping at the receptor homodimer interface, are likely critical for functional recognition of the m157 glycoprotein. Our combined functional and three-dimensional modeling approach suggested that the architecture of the Ly49H dimer is crucial to accessing m157, but not MHC class I. These results link Ly49 homodimerization variability to the direct recognition of pathogen products.     

Characterization of murine cytomegalovirus m157 from infected cells and identification of critical residues mediating recognition by the NK cell receptor Ly49H

Activated NK cells mediate potent cytolytic and secretory effector functions and are vital components of the early antiviral immune response. NK cell activities are regulated by the assortment of inhibitory receptors that recognize MHC class I ligands expressed on healthy cells and activating receptors that recognize inducible host ligands or ligands that are not well characterized. The activating Ly49H receptor of mouse NK cells is unique in that it specifically recognizes a virally encoded ligand, the m157 glycoprotein of murine CMV (MCMV). The Ly49H-m157 interaction underlies a potent resistance mechanism (Cmv1) in C57BL/6 mice and serves as an excellent model in which to understand how NK cells are specifically activated in vivo, as similar receptor systems are operative for human NK cells. For transduced cells expressing m157 in isolation and for MCMV-infected cells, we show that m157 is expressed in multiple isoforms with marked differences in abundance between infected fibroblasts (high) and macrophages (low). At the cell surface, m157 is exclusively a glycosylphosphatidylinositol-associated protein in MCMV-infected cells. Through random and site-directed mutagenesis of m157, we identify unique residues that provide for efficient cell surface expression of m157 but fail to activate Ly49H-expressing reporter cells. These m157 mutations are predicted to alter the conformation of a putative m157 interface with Ly49H, one that relies on the position of a critical alpha0 helix of m157. These findings support an emerging model for a novel interaction between this important NK cell receptor and its viral ligand.     

Composition,Antigenic Properties and Hepatocyte Surface Expression of the Woodchuck Asialoglycoprotein Receptor

Abstract

We have purified woodchuck hepatic asialoglycoprotein receptor (ASGPR) by ligand affinity chromatography and have identified it as a heterooligomeric complex comprised of two subunits with molecular masses of 40 and 47 kD, designated as woodchuck hepatic lectin 1 and 2 (WHL1 and WHL2), respectively. With the help of antisera generated against the soluble, bioactive woodchuck and rabbit ASGPRs and anti-subunit monospecific antibodies, distinct antigenic specificity of each of the ASGPR polypeptide subunits and interspecies immunologic cross-reactivity of the receptor polypeptides displaying comparable molecular masses were documented. In contrast to the purified woodchuck receptor, WHL2 antigenic reactivity was not identifiable in woodchuck hepatocyte plasma membranes unless the intact membranes were exposed to an asialylated ligand or a soluble membrane fraction was incubated with anti-receptor antibody. These findings imply that both WHL1 and WHL2 are expressed on the hepatocyte surface and contribute to ligand binding, since antibody specific to either subunit blocks ligand attachment. Our results also indicate that ligand binding modifies antigenic properties of the membrane expressed ASGPR.     

Structural and functional aspects of the Ly49 natural killer cell receptors

Natural killer cells are part of the first line of innate immune defence against virus-infected cells and cancer cells in the vertebrate immune system. They are called 'natural' killers because, unlike cytotoxic T cells, they do not require a previous challenge and preactivation to become active. The Ly49 NK receptors are type II transmembrane glycoproteins, structurally characterized as disulphide-linked homodimers. They share extensive homology with C-type lectins, and they are encoded by a multigene family that in mice maps on chromosome 6. A fine balance between inhibitory and activating signals regulates the function of NK cells. Inhibitory Ly49 molecules bind primarily MHC class I ligands, whereas the ligands for activating Ly49 molecules may include MHC class I, but also interestingly MHC class I-like molecules expressed by viruses, as is the case for Ly49H, which binds the m157 gene product of murine cytomegalovirus. In this study, we review the function and X-ray crystal structure of the Ly49 NK cell receptors hitherto determined (Ly49A, Ly49C and Ly49I), and the structural features of the Ly49/MHC class I interaction as revealed by the X-ray crystal structures of Ly49A/H-2Dd and the recently determined Ly49C/H-2Kb.     

Contribution of the Ly49E Natural Killer Receptor in the Immune Response to Plasmodium berghei Infection and Control of Hepatic Parasite Development

Natural killer (NK) cells have different roles in the host response against Plasmodium-induced malaria depending on the stage of infection. Liver NK cells have a protective role during the initial hepatic stage of infection by production of the T_H1-type cytokines IFN-γ and TNF-α. In the subsequent erythrocytic stage of infection, NK cells also induce protection through Th1-type cytokines but, in addition, may also promote development of cerebral malaria via CXCR3-induction on CD8⁺ T cells resulting in migration of these cells to the brain. We have recently shown that the regulatory Ly49E NK receptor is expressed on liver NK cells in particular. The main objective of this study was therefore to examine the role of Ly49E expression in the immune response upon Plasmodium berghei ANKA infection, for which we compared wild type (WT) to Ly49E knockout (KO) mice. We show that the parasitemia was higher at the early stage, i.e. at days 6–7 of Plasmodium berghei ANKA infection in Ly49E KO mice, which correlated with lower induction of CD69, IFN-γ and TNF-α in DX5⁻ liver NK cells at day 5 post-infection. At later stages, these differences faded. There was also no difference in the kinetics and the percentage of cerebral malaria development and in lymphocyte CXCR3 expression in WT versus Ly49E KO mice. Collectively, we show that the immune response against Plasmodium berghei ANKA infection is not drastically affected in Ly49E KO mice. Although NK cells play a crucial role in Plasmodium infection and Ly49E is highly expressed on liver NK cells, the Ly49E NK receptor only has a temporarily role in the immune control of this parasite.     

The Urokinase Receptor Takes Control of Cell Migration by Recruiting Integrins and FPR1 on the Cell Surface

The receptor (uPAR) of the urokinase-type plasminogen activator (uPA) is crucial in cell migration since it concentrates uPA proteolytic activity at the cell surface, binds vitronectin and associates to integrins. uPAR cross-talk with receptors for the formylated peptide fMLF (fMLF-Rs) has been reported; however, cell-surface uPAR association to fMLF-Rs on the cell membrane has never been explored in detail.We now show that uPAR co-localizes at the cell-surface and co-immunoprecipitates with the high-affinity fMLF-R, FPR1, in uPAR-transfected HEK-293 (uPAR-293) cells. uPAR/β1 integrin and FPR1/β1 integrin co-localization was also observed. Serum or the WKYMVm peptide (W Pep), a FPR1 ligand, strongly increased all observed co-localizations in uPAR-293 cells, including FPR1/β1 integrin co-localization. By contrast, a low FPR1/β1 integrin co-localization was observed in uPAR-negative vector-transfected HEK-293 (V-293) cells, that was not increased by serum or W Pep stimulations.The role of uPAR interactions in cell migration was then explored. Both uPAR-293 and V-293 control cells efficiently migrated toward serum or purified EGF. However, cell treatments impairing uPAR interactions with fMLF-Rs or integrins, or inhibiting specific cell-signaling mediators abrogated uPAR-293 cell migration, without exerting any effect on V-293 control cells.Accordingly, uPAR depletion by a uPAR-targeting siRNA or uPAR blocking with an anti-uPAR polyclonal antibody in cells constitutively expressing high uPAR levels totally impaired their migration toward serum.Altogether, these results suggest that both uPAR-positive and uPAR-negative cells are able to migrate toward serum; however, uPAR expression renders cell migration totally and irreversibly uPAR-dependent, since it is completely inhibited by uPAR blocking.We propose that uPAR takes control of cell migration by recruiting fMLF-Rs and β1 integrins, thus promoting their co-localization at the cell-surface and driving pro-migratory signaling pathways.     

Inhibitory NK Receptor Recognition of HLA-G: Regulation by Contact Residues and by Cell Specific Expression at the Fetal-Maternal Interface

The non-classical HLA-G protein is distinguished from the classical MHC class I molecules by its expression pattern, low polymorphism and its ability to form complexes on the cell surface. The special role of HLA-G in the maternal-fetal interface has been attributed to its ability to interact with specific receptors found on maternal immune cells. However this interaction is restricted to a limited number of receptors. In this study we elucidate the reason for this phenomenon by comparing the specific contact residues responsible for MHC-KIR interactions. This alignment revealed a marked difference between the HLA-G molecule and other MHC class I molecules. By mutating these residues to the equivalent classical MHC residues, the HLA-G molecule regained an ability of interacting with KIR inhibitory receptors found on NK cells derived either from peripheral blood or from the decidua. Functional NK killing assays further substantiated the binding results. Furthermore, double immunofluorescent staining of placental sections revealed that while the conformed form of HLA-G was expressed in all extravillous trophoblasts, the free heavy chain form of HLA-G was expressed in more distal cells of the column, the invasion front. Overall we suggest that HLA-G protein evolved to interact with only some of the NK inhibitory receptors thus allowing a control of inhibition, while permitting appropriate NK cell cytokine and growth factor production necessary for a viable maternal fetal interface.     

Efficient Transduction by an Amphotropic Retrovirus Vector Is Dependent on High-Level Expression of the Cell Surface Virus Receptor

Transduction by murine leukemia virus-based retrovirus vectors is limited in certain cell types, particularly in nondividing cells. But transduction can be inefficient even in cells that divide rapidly. For example, exposure of 208F rat embryo fibroblasts to an excess of an amphotropic retrovirus vector encoding alkaline phosphatase results in a transduction efficiency of only about 10%, even though these cells divide rapidly. Here we show that transduction of 208F cells is limited by cell surface retrovirus receptor levels; overexpression of the amphotropic retrovirus receptor Pit2 markedly improved the transduction efficiency to 50%. To characterize receptor levels and binding affinity, we synthesized a fusion protein that joins the amino terminus of the amphotropic envelope protein to the Fc region of a human immunoglobulin G1 molecule for use in binding assays. In comparison to the parental cell line, the modified cell line showed an order of magnitude increase in binding sites of from 18,000 to 150,000 per cell. Thus, efficient transduction by an amphotropic retrovirus vector requires high-level expression of the retrovirus receptor Pit2. These results provide the rationale for further examination of the role of receptor levels in inefficient transduction, especially with regard to target cells for gene therapy, where a high transduction rate is often crucial.     

Characterisation of Curli Production, Cell Surface Hydrophobicity, Autoaggregation and Attachment Behaviour of Escherichia coli O157

Escherichia coli O157 are an important group of foodborne pathogens with the ability to attach to materials commonly used in food processing environments such as slightly hydrophilic stainless steel. The aim of this study was to characterise six E. coli isolates, including five E. coli O157, for curli production, autoaggregation, hydrophobicity and attachment to highly hydrophilic glass and hydrophobic Teflon^®. Curli production and autoaggregation were determined using absorbance assays; hydrophobicity by bacterial adherence to hydrocarbons, hydrophobic interaction chromatography and contact angle measurements; and attachment using epifluorescence microscopy. Curli production varied between strains and for some strains correlated with autoaggregation. Curli production correlated with decreased hydrophobicity for two strains. Four of the six isolates increased attachment to glass, but decreased attachment to Teflon^® with increased curli production. In contrast, one of the six isolates decreased attachment to glass, but increased attachment to Teflon^® with increasing curli production. Curli production by the remaining isolate did not correlate with hydrophobicity or attachment. Attachment of some E. coli, including E. coli O157, to abiotic surfaces may be influenced by curli production, autoaggregation and hydrophobicity. However, for other strains, a variety of factors may be of greater influence on these properties and ability to attach to abiotic surfaces. This study highlights the complexity of bacterial surface properties and their relationship with bacterial attachment.     

Tolerance and alloreactivity of the Ly49D subset of murine NK cells.

Class I-specific stimulatory and inhibitory receptors expressed by NK cell subsets contribute to the alloreactive potential of the self-tolerant murine NK cell repertoire. In this report, we have studied potential mechanisms of tolerance to the function of the positive signaling Ly49D receptor in mice that express one of its ligands, H2-Dd. Our results demonstrate that H2-Dd-expressing mice possess a large Ly49D+ subset of NK cells that is functionally capable of rejecting bone marrow cell (BMC) allografts in vivo and lysing allogeneic Con A lymphoblasts in vitro. Also, we show that the Ly49D receptor is responsible for the ability of H2b/d F1 hybrid mice to reject H2d/d parental BMC (hybrid resistance). Thus, deletion or anergy of Ly49D+ cells in H2-Dd+ hosts cannot explain self tolerance. Our functional studies revealed that coexpression of the Dd-specific Ly49A or Ly49G2 inhibitory receptors by Ly49D+ cells resulted in tolerance to Dd+ targets, while coexpression of Kb-specific inhibitory receptors Ly49C/I resulted in tolerance to Kb+ targets. Only in H2d/d cells did Ly49C/I dominantly inhibit Ly49D-Dd stimulation. This correlated with an increased mean fluorescence intensity of Ly49C expression, as well as an increased percentage of Ly49C+ cells in the Ly49D+A/G2- compartment. Therefore, we conclude that self tolerance of the Ly49D subset can be achieved through coexpression of a sufficient level of self-specific inhibitory receptors.     

The Cell Surface Receptor Tartan Is a Potential In Vivo Substrate for the Receptor Tyrosine Phosphatase Ptp52F

Receptor-linked protein-tyrosine phosphatases (RPTPs) are essential regulators of axon guidance and synaptogenesis in Drosophila, but the signaling pathways in which they function are poorly defined. We identified the cell surface receptor Tartan (Trn) as a candidate substrate for the neuronal RPTP Ptp52F by using a modified two-hybrid screen with a substrate-trapping mutant of Ptp52F as “bait.” Trn can bind to the Ptp52F substrate-trapping mutant in transfected Drosophila S2 cells if v-Src kinase, which phosphorylates Trn, is also expressed. Coexpression of wild-type Ptp52F causes dephosphorylation of v-Src-phosphorylated Trn. To examine the specificity of the interaction in vitro, we incubated Ptp52F-glutathione S-transferase (GST) fusion proteins with pervanadate-treated S2 cell lysates. Wild-type Ptp52F dephosphorylated Trn, as well as most other bands in the lysate. GST “pulldown” experiments demonstrated that the Ptp52F substrate-trapping mutant binds exclusively to phospho-Trn. Wild-type Ptp52F pulled down dephosphorylated Trn, suggesting that it forms a stable Ptp52F-Trn complex that persists after substrate dephosphorylation. To evaluate whether Trn and Ptp52F are part of the same pathway in vivo, we examined motor axon guidance in mutant embryos. trn and Ptp52F mutations produce identical phenotypes affecting the SNa motor nerve. The genes also display dosage-dependent interactions, suggesting that Ptp52F regulates Trn signaling in SNa motor neurons.Receptor-linked protein-tyrosine phosphatases (RPTPs) are enzymes with extracellular (XC) domains, a single transmembrane domain, and one or two cytoplasmic protein tyrosine phosphatase (PTP) homology domains. Many RPTPs have XC sequences that resemble those of cell adhesion molecules (for a review, see reference 33). This sequence organization suggests that RPTPs can couple cell-cell recognition events to dephosphorylation of cytoplasmic substrates. Interestingly, while phosphotyrosine (PY) pathways involved in cell growth and differentiation typically involve receptor tyrosine kinases that bind to growth factors and are regulated by nontransmembrane PTPs, those that control axon guidance often use RPTPs and nontransmembrane TKs. This implies that the cues that affect PY signaling in axonal growth cones may interact with RPTPs rather than with receptor tyrosine kinases (reviewed in reference 14).There are 17 active RPTPs encoded in the human genome, while Drosophila has six. Most of the mammalian RPTPs are expressed in nonneural tissues, but four of the six fly RPTPs are expressed only by central nervous system (CNS) neurons in late embryos. All published zygotic phenotypes produced by Rptp mutations are alterations in axon guidance or synaptogenesis. These results suggest that the major functions of the Drosophila RPTPs are in neural development (for a review, see reference 16). Analysis of axon guidance phenotypes in embryos bearing single or multiple Rptp mutations is consistent with the idea that RPTP interactions with ligands at growth cone choice points convey “information,” in the form of changes in substrate phosphorylation within growth cones, that is used to determine pathway decisions.In the Drosophila neuromuscular system, 36 motor axons grow out within six nerve bundles in each abdominal hemisegment, and each axonal growth cone makes a series of genetically determined guidance decisions that direct it to the appropriate muscle fiber (for a review, see reference 27). Our work on Rptp mutant combinations suggests that each pathway decision uses a specific subset of the six RPTPs. RPTPs can exhibit functional redundancy, so that the loss of one does not produce a defect unless another RPTP is also absent, or competition, in which loss of one RPTP suppresses the phenotype produced by loss of another (5, 6, 31). Examination of RPTP expression patterns suggests that the RPTPs are expressed by most (or possibly all) CNS neurons, including motor neurons. If so, the requirements for individual RPTPs for execution of particular guidance decisions cannot be due to selective expression of these RPTPs on specific motor axons. These requirements might instead be determined by the expression patterns of RPTP ligands, so that only RPTPs whose ligands were localized to the vicinity of a growth cone choice point would participate in that pathway decision. Alternatively (or in addition), the necessity of a particular RPTP for a pathway decision might arise from selective expression of RPTP substrates, so that an RPTP would be important for guidance decisions made by a growth cone of a specific motor neuron only if that neuron expressed the relevant substrate(s).Evaluation of such models requires identification of specific XC ligands and intracellular substrates for the Drosophila RPTPs. Only one set of ligands has been identified thus far. These are the heparan sulfate proteoglycans Syndecan (Sdc) and Dallylike (Dlp), which bind to the Lar RPTP with nanomolar affinity and contribute to its functions in axon guidance and synapse growth (9, 15). Similarly, little is known about substrate specificity in vivo. Lar can dephosphorylate the Enabled (Ena) protein, which regulates the growth cone cytoskeleton, and genetic interaction studies suggest that Ena may be an in vivo substrate for Lar (35). The transmembrane protein gp150 can be dephosphorylated by Ptp10D in cell culture and intact fly larvae, but genetics has not provided evidence that Ptp10D and gp150 are in the same signaling pathway in vivo (7).The identification of in vivo substrates for RPTPs has been hampered by the fact that purified RPTP cytoplasmic domains often do not exhibit high selectivity in vitro when tested for dephosphorylation activity on peptides or proteins. The most fruitful method for finding substrates for both RPTPs and cytoplasmic PTPs has been the use of “substrate-trapping” mutants. The most effective substrate traps were devised by Tonks and coworkers, and are created by changing an invariant Asp (D) residue within the PTP active site to Ala (A) (8). The D residue has an abnormal pK and is thus able to donate a proton to the phosphorus-oxygen bond, facilitating displacement of the tyrosine (Y) OH by the invariant Cys (C) nucleophile of the enzyme. This creates a phosphoenzyme intermediate. The dephosphorylated substrate then dissociates, and water attacks the Cys-phosphate bond, releasing the phosphate and reconstituting the enzyme. In D→A mutants, the polarization of the phosphorus-oxygen bond by protonation cannot take place, and the PY substrate remains bound to the enzyme. Substrate-trapping mutants expressed in cells often bind to only a few phosphoproteins, suggesting that PTPs exhibit high specificity in vivo (see, for example, reference 11).We conducted a modified yeast two-hybrid screen to find Drosophila phosphoproteins that bind selectively to RPTP substrate-trapping mutants. We identified the cell surface receptor Tartan (Trn) in this screen and showed that it is a substrate for the Ptp52F RPTP in Drosophila Schneider 2 (S2) cells. Axon guidance phenotypes in trn mutants are identical to those seen in Ptp52F mutants, and trn and Ptp52F exhibit dosage-dependent genetic interactions. These results suggest that Ptp52F is a regulator of Trn signaling in motor neurons in vivo.     

RACK1 Regulates the Cell Surface Expression of the G Protein-Coupled Receptor for Thromboxane A2

We used the yeast two-hybrid system to screen for proteins that interact with the C-terminus of the β isoform of the thromboxane A₂ receptor (TPβ). This screen identified receptor for activated C-kinase 1 (RACK1) as a new TPβ-interacting protein. Here, we show that RACK1 directly binds to the C-terminus and the first intracellular loop of TPβ. The TPβ–RACK1 association was further confirmed by co-immunoprecipitation studies in HEK293 cells and was not modulated by stimulation of the receptor. We observed that cell surface expression of TPβ was increased when RACK1 was overexpressed, while it was inhibited when endogenous RACK1 expression was knocked down by small interfering RNA. Confocal microscopy confirmed the impaired cell surface expression of TPβ and suggested that the receptors remained predominantly localized in the endoplasmic reticulum (ER) in RACK1-depleted cells. Confocal microscopy also revealed that a transient TPβ–RACK1 association takes place in the ER. The effect of RACK1 on receptor trafficking to the cell surface appears to be selective to some G protein-coupled receptors (GPCRs) because inhibition of RACK1 expression also affected cell surface targeting of the angiotensin II type 1 receptor and CXCR4 but not of β₂-adrenergic and prostanoid DP receptors. Our data demonstrate for the first time a direct interaction between RACK1 and a GPCR and identify a novel role for RACK1 in the regulation of the transport of a membrane receptor from the ER to the cell surface.     

N-Linked Oligosaccharides Are Required for Cell Surface Expression of the Norepinephrine Transporter but Do Not Influence Substrate or Inhibitor Recognition

Abstract: The contribution of N-linked carbohydrates to the function of the human norepinephrine transporter (NET) was investigated using site-directed mutagenesis to inactivate the two most carboxy-terminal (NQQ mutant) or all three (QQQ mutant) sites for N -glycosylation within the extracellular loop between transmembrane domains 3 and 4. In HeLa cells transiently expressing the NET, two glycosylated forms of the transporter at 90 and 60 kDa are immunoprecipitated by NET antisera. A single 50-kDa species is observed in cells expressing the QQQ mutant, and it likely represents the NET core protein. Analyses of substrate transport kinetics showed rank order V _max of 19:9:1 for NET/NQQ/QQQ without a change in the apparent affinity of the wild-type and mutated carriers for either substrates or transport inhibitors. Cell surface biotinylation indicates that all NET, NQQ, and QQQ transporter species are detected at the plasma membrane but that glycosylated forms are selectively enriched. The transport activities exhibited by each of the carriers correlate well with cell surface content. Subcellular localization of transporters using immunofluorescence microscopy shows that reductions in surface expression and transport are associated with a corresponding increase in the intracellular retention of mutated carriers. Thus, N-linked glycosylation does not alter the apparent affinity of NET for either substrates or inhibitors of transport but, instead, appears to influence the abundance of carriers at the cell surface.