Cell Receptor Interactions Research Articles

For T Cell Receptors, Some Breakups Might Not Last Forever

Does the affinity or half-life of peptide-MHC-T cell receptor (TCR) interactions determine T cell activation? In this issue of Immunity, Aleksic et al. (2010) propose a role for the on rate through multiple rebindings to the same TCR.

Quantitative Measurements of Receptor Interactions in Mammalian Cells: Implications for Human Pathologies

Receptor Tyrosine kinases (RTKs) are family of single-pass cell membrane receptors with extracellular ligand-binding domains and intracellular kinase domains, which conduct biochemical signals via lateral dimerization in the plasma membrane. Mutations in the transmembrane (TM) domains of these receptors are known to promote unregulated signaling. An example is the Ala391Glu mutation in the TM domain of FGFR3, which leads to pathologies such as Crouzon syndrome and bladder cancer. This mutation has been shown to stabilize the isolated TM domain dimers in lipid membranes, but it is not known if it stabilizes the full length FGFR3 receptor dimers in the plasma membrane of mammalian cells. To address the effect of the mutation in mammalian cells, we have determined free energies of dimerization for the wild type and mutant FGFR3 in mammalian (HEK293T and CHO) plasma membranes using the QI Förster resonance energy transfer (FRET) technique [Li et al.,2008]. The measured change in the dimerization free energy due to the Ala391Glu mutation is −1.2 kcal/mol, consistent with previous reports of hydrogen bond strength in proteins, as well as results for the isolated FGFR3 TM domains. Thus, we have shown that the mutation stabilizes the full length FGFR3 dimers in mammalian cells. We propose that this dimer stabilization is the major cause for FGFR3 overactivation and human pathologies. Li E,Placone J,Merzlyakov M, Hristova K (2008) Quantitative measurements of protein interactions in a crowded cellular environment.Anal Chem 80:5976-5985.

The nonpolymorphic MHC Qa-1b mediates CD8+ T cell surveillance of antigen-processing defects

The nonclassical major histocompatibility complex (MHC) Qa-1b accommodates monomorphic leader peptides and functions as a ligand for germ line receptors CD94/NKG2, which are expressed by natural killer cells and CD8+ T cells. We here describe that the conserved peptides are replaced by a novel peptide repertoire of surprising diversity as a result of impairments in the antigen-processing pathway. This novel peptide repertoire represents immunogenic neoantigens for CD8+ T cells, as we found that these Qa-1b–restricted T cells dominantly participated in the response to tumors with processing deficiencies. A surprisingly wide spectrum of target cells, irrespective of transformation status, MHC background, or type of processing deficiency, was recognized by this T cell subset, complying with the conserved nature of Qa-1b. Target cell recognition depended on T cell receptor and Qa-1b interaction, and immunization with identified peptide epitopes demonstrated in vivo priming of CD8+ T cells. Our data reveal that Qa-1b, and most likely its human homologue human leukocyte antigen-E, is important for the defense against processing-deficient cells by displacing the monomorphic leader peptides, which relieves the inhibition through CD94/NKG2A on lymphocytes, and by presenting a novel repertoire of immunogenic peptides, which recruits a subset of cytotoxic CD8+ T cells.

Measurement of bluetongue virus binding to a mammalian cell surface receptor by an in situ immune fluorescent staining technique

A quantifiable in situ immune fluorescent assay (IFA) was developed to measure bluetongue virus (BTV) binding to mammalian cells. The utility of the assay was demonstrated with both Chinese hamster ovary (CHO) and bovine pulmonary artery endothelial (CPAE) cells. Since heparin sulfate (HS) has been shown to function as a receptor for a number of viruses, its role as a receptor for BTV was evaluated with the in situ IFA. Binding of BTV to both CHO and CPAE cells was inhibited in a dose dependent manner by HS. In addition, HS deficient CHO cells showed greatly diminished binding of BTV when compared to the parental cell line. The IFA protocol will find application, as a non-isotopic, quantifiable technique, to study virus–cell receptor interactions. Information gained from such studies will expand our understanding of the early steps in virus replication.

Structure of yellow fever virus envelope protein domain III

The structure of recombinant domain III of the envelope protein (rED3) of yellow fever virus (YFV), containing the major neutralization site, was determined using NMR spectroscopy. The amino acid sequence and structure of the YFV-rED3 shows differences from ED3s of other mosquito-borne flaviviruses; in particular, the partially surface-exposed BC loop where methionine-304 and valine-324 were identified as being critical for the structure of the loop. Variations in the structure and surface chemistry of ED3 between flaviviruses affect neutralization sites and may affect host cell receptor interactions and play a role in the observed variations in viral pathogenesis and tissue tropism.

Hyaluronan receptors involved in cytokine induction in monocytes

During inflammation, lower molecular weight fragments of hyaluronan accumulate, and this is known to be inflammatory and immune-stimulatory. In diseases such as inflammatory bowel disease, inflammatory cells bind to hyaluronan; however, the cellular response and molecular mechanism of hyaluronan-hyaluronan receptor interactions in mononuclear cells are not well understood. The expression of hyaluronan receptors in peripheral blood mononuclear cells (PBMC) was examined. PBMC were stimulated with lower and higher molecular weight hyaluronan (molecular weight 100-150 kDa and 2700 kDa) and the induction of proinflammatory cytokines (interleukin-6 (IL-6) and monocyte chemoattractant protein (MCP-1)) was compared by enzyme-linked immunoabsorbant assay (ELISA). Cells were coincubated with various signaling pathway inhibitors. In addition, neutralizing antibodies against CD44 and TLR4 were added and the effects on PBMC were investigated. Finally, mononuclear cells from CD44-null and toll-like receptor 4 (TLR4) mutant mice were both stimulated with lower molecular weight hyaluronan. Among the hyaluronan receptors, TLR4 and CD44 were markedly expressed on PBMC. Hyaluronan-stimulated PBMC enhanced the attachment to the extracellular matrix. Lower molecular weight hyaluronan induced IL-6 and MCP-1 production in PBMC, but high-molecular-weight hyaluronan did not induce IL-6 and MCP-1 production. An anti-CD44 antibody attenuated the induction of both IL-6 and MCP-1 in lower molecular weight hyaluronan-stimulated PBMC. In both TLR4 mutant and CD44-null mice, the induction of IL-6 by lower molecular weight hyaluronan stimulation was decreased. SB203580 completely abolished IL-6 production in both TLR4 mutant and CD44-null mononuclear cells, while PD98059 abolished IL-6 production in CD44-null mononuclear cells. Hyaluronan receptors, CD44 and TLR4, play distinct roles in cytokine induction in hyaluronan-stimulated mononuclear cells.

Crossreactive T Cells Spotlight the Germline Rules for αβ T Cell-Receptor Interactions with MHC Molecules

To test whether highly crossreactive alphabeta T cell receptors (TCRs) produced during limited negative selection best illustrate evolutionarily conserved interactions between TCR and major histocompatibility complex (MHC) molecules, we solved the structures of three TCRs bound to the same MHC II peptide (IAb-3K). The TCRs had similar affinities for IAb-3K but varied from noncrossreactive to extremely crossreactive with other peptides and MHCs. Crossreactivity correlated with a shrinking, increasingly hydrophobic TCR-ligand interface, involving fewer TCR amino acids. A few CDR1 and CDR2 amino acids dominated the most crossreactive TCR interface with MHC, including Vbeta8 48Y and 54E and Valpha4 29Y, arranged to impose the familiar diagonal orientation of TCR on MHC. These interactions contribute to MHC binding by other TCRs using related V regions, but not usually so dominantly. These data show that crossreactive TCRs can spotlight the evolutionarily conserved features of TCR-MHC interactions and that these interactions impose the diagonal docking of TCRs on MHC.

Cell receptor interaction chromatography: importance of corrosion processes

Corrosion is a term traditionally describing degradation processes in metals. Modern dictionaries explain corrosion as: 'erosion by chemical action' or 'a state of deterioration in metals caused by oxidation or chemical action'. Erosion by chemical action applies to degradation processes in both metals and polymers, with humidity as one principal actor. In polymers, extended humidity exposure can result in manifest corrosion. Less clear is the action of transient humidity exposure, causing corrosion, or reversible effects mistakenly interpreted as corrosion. Effects of transient humidity exposure on polymers have been recently investigated by contact angle measurements. However, contact angles are insensitive to various parameters, including changes in topography. In this paper the author illustrates the importance of minimal effects of corrosion in polymers, exemplary for the method of cell receptor interaction chromatography (CRIC), a method initially designed to predict specific biocompatibility in an aqueous milieu. The method of CRIC exploits the interplay between the attachment of nanospheres suspended in drops to nanostructured substrates and their transport by convection, and discriminates between physical and chemical parameters involved in corrosion.

Killer Immunoglobulin Receptor (KIR) Negative NK Cells Exhibit Potent Alloreactivity Against Primary Leukemia Targets When Interrupting MHC Class I Interactions through NKG2A Blockade.

Effective therapeutic options are lacking for patients with primary refractory or relapsed acute myelogenous leukemia (AML). We have shown that adoptive transfer of haploidentical NK cells can result in complete remissions (25%) in patients with refractory disease but this procedure is still limited by a high failure rate. Based on the finding that 29±3% (n=26) of normal CD56dim NK cells are KIR− NKG2A+, we hypothesized that non-KIR class I MHC inhibitory receptors may play a bigger role than previously recognized in the cumulative integration of signals that determine whether leukemia targets are killed by NK cells. We studied the role of NKG2A and KIR inhibitory signals on primary AML and acute lymphoblastic leukemia (ALL) targets collected by therapeutic lymphapheresis. Blast susceptibility to fresh resting polyclonal allogeneic NK cells (enriched by CD3 depletion) and to NK cells activated with low pharmacologic doses of IL-2 for 72 hours was determined using flow cytometry based cytotoxicity assays and a degranulation assay using CD107a. The role of inhibitory signaling through class I MHC-recognizing receptors was tested by blocking interactions between receptor and cognate ligand using 1) a pan-MHC monoclonal antibody (mAb) (clone HP17-F) recognizing HLA-A, B, C and E, 2) an anti-KIR reagent (1-7F9 which blocks inhibitory KIR2DL1/L2/L3) currently in clinical trials (Novonordisk, Copenhagen) and 3) a mAb against NKG2A (clone Z199). Susceptibility to killing was defined as >10% lysis at an E:T ratio of 10:1. Two of 8 leukemias (25%) were lysed by resting allogeneic NK cells. NK cells activated with IL-2 (5U/ml) killed 7 of 8 targets (average increase of 13±2.2% lysis). In 3 AMLs killing was significantly enhanced by pan-MHC mAb blockade, but was less enhanced by anti-KIR blockade. NKG2A blockade alone also increased killing, and when added to anti-KIR blockade AML killing was equal to that obtained with MHC blockade. This suggests that non-KIR class I recognizing interactions were operant. Interestingly, anti-KIR blockade did significantly enhance killing of ALL targets suggesting that higher MHC expression (MFI 3-fold greater) may be a dominant factor to NK cell susceptibility in ALL. These findings support the notion that susceptibility to NK cell lysis is determined by interactions beyond just KIR and KIR-ligands. To explore this further KIR− NK cells were enriched with immunomagnetic bead separation (80% NKG2A+). AML target killing by these IL-2 activated KIR−NKG2A+ NK cells increased significantly after pan-MHC and NKG2A blockade (from an average baseline of 21% lysis to 34% for both), but no increase was seen with KIR blockade. This suggests that a significant proportion of normal NK cells are negatively regulated by KIR-independent mechanisms interacting with HLA-E. In summary, interrupting dominant NK cell receptor interactions with class I MHC ligands may lead to better strategies to treat AML and even ALL. NKG2A blockade may be a good target as it is highly expressed on virtually all NK cells reconstituting in the first 3 months after allogeneic HCT and it identifies NK cells with effector function in normal subjects (Cooley et al, Blood 110:578 2007). The combination of inhibitory signal blockade with other activators such as low dose IL-2 or mAb capable of ADCC may be needed to best exploit the clinical therapeutic potential of NK cells.

CD40 ligand and MHC class II expression are essential for human peripheral B cell tolerance

Hyper-IgM (HIGM) syndromes are primary immunodeficiencies characterized by defects of class switch recombination and somatic hypermutation. HIGM patients who carry mutations in the CD40-ligand (CD40L) gene expressed by CD4+ T cells suffer from recurrent infections and often develop autoimmune disorders. To investigate the impact of CD40L–CD40 interactions on human B cell tolerance, we tested by ELISA the reactivity of recombinant antibodies isolated from single B cells from three CD40L-deficient patients. Antibody characteristics and reactivity from CD40L-deficient new emigrant B cells were similar to those from healthy donors, suggesting that CD40L–CD40 interactions do not regulate central B cell tolerance. In contrast, mature naive B cells from CD40L-deficient patients expressed a high proportion of autoreactive antibodies, including antinuclear antibodies. Thus, CD40L–CD40 interactions are essential for peripheral B cell tolerance. In addition, a patient with the bare lymphocyte syndrome who could not express MHC class II molecules failed to counterselect autoreactive mature naive B cells, suggesting that peripheral B cell tolerance also depends on major histocompatibility complex (MHC) class II–T cell receptor (TCR) interactions. The decreased frequency of MHC class II–restricted CD4+ regulatory T cells in CD40L-deficient patients suggests that these T cells may mediate peripheral B cell tolerance through CD40L–CD40 and MHC class II–TCR interactions.

Interaction between α1 and α2 adrenergic receptors in mice mesenteric veins and HEK293 cells

Norpinephrine (NE) released by sympathetic nerves constricts arteries and veins. Veins are more sensitive to NE than arteries and veins, but not arteries, are resistant to desensitization caused by NE. This might occur because α2 adrenergic receptors (AR) indirectly contribute to NE constrictions of veins, but not arteries. α1DAR and α2CAR mediate NE-induced venous contraction. Therefore, we studied receptor interactions in HEK-293 cells expressing α1DAR and α2CAR. Immuocytochemistry revealed that α1DAR were localized intracellularly but co-expression with α2CAR did not alter this cellular localization. Similar results were obtained when α1DAR was co-expressed with α2AAR and α2BAR. Co-expression with β2AR enhanced membrane expression of α1DAR. Functional interactions were investigated using calcium imaging techniques. NE caused an increase in calcium in α1DAR expressing cells, these responses were not altered by co-expression with any α2-AR. Co-expression of β2AR with α1DAR caused an increase in NE-induced calcium signals. α1DAR desensitization caused by NE was not altered by co-expression with α2ARs. These findings suggest, that α1D and α2AR co-expression alone does not account for enhanced NE sensitivity and resistance to desensitization in veins compared to arteries. Intracellular signaling mechanisms might contribute to artery-vein differences in adrenergic reactivity.

Altered Peptide Ligands Induce Delayed CD8-T Cell Receptor Interaction—a Role for CD8 in Distinguishing Antigen Quality

How T cells translate T cell receptor (TCR) recognition of almost identical pMHC ligands into distinct biological responses has remained enigmatic. Although differences in affinity or off rate are important, they offer at best an incomplete explanation. By using Förster resonance energy transfer (FRET), we have visualized the ligand-induced interaction between OT-I TCR and CD8. We found that both recruitment of TCR to the immunological synapse and the TCR-CD8 interaction induced by weak agonists (positive-selecting ligands) was delayed but not necessarily weaker than strong agonists (negative selectors). A delayed and perhaps longer lasting CD8-TCR interaction results in delayed phospho-ERK recruitment to the synapse. The kinetics of the TCR-CD8 interaction can reconcile previously anomalous data, where biological activity did not correlate with TCR-pMHC binding kinetics for certain ligands. Our findings indicate that the T cell translates antigen recognition into T cell responses by differential recruitment of CD8 to the TCR.

Probing drug-cell interactions

Over the past two decades, the subset of scanning probe microscopy termed atomic force microscopy (AFM) has become a ubiquitous tool to image nanoscale structure and to estimate certain mechanical characteristics of biological entities ranging from DNA to tissues. Various modes of imaging and force spectroscopies have been developed to correlate structure, properties, and chemomechanical interactions of molecules and cells in aqueous environments. These advances in AFM have led rapidly to in situ investigations of drug-induced changes in cell structure, membrane stability, and receptor interaction forces.

Detection and Characterization of Avian Leukosis Virus in Marek's Disease Vaccines

Avian leukosis virus (ALV) infection in chickens is known to induce increased mortality, tumors, delayed growth, and suboptimal egg production. Countries importing specified pathogen-free eggs, vaccines, and poultry breeding stock require freedom of infection or contamination with ALV in such products among other avian pathogens. Recently, ALV was found as a contaminant in a limited number of commercial poultry vaccines, even after routine quality assurance procedures cleared the vaccines for commercialization. The contaminated vaccines were promptly withdrawn from the market, and no direct detrimental effects were reported in poultry vaccinated with such vaccines. We describe herein the characterization in vitro of the contaminant viruses. All exogenous viruses detected in four vaccine lots belong to subgroup A of ALV based on cell receptor interaction, subgroup-specific polymerase chain reaction (PCR), envelope gene sequencing, and virus neutralization. A combination of thermal treatment and serial dilutions of the contaminated vaccines facilitated detection of contaminating ALVs in cell culture coupled with antigen-capture enzyme-linked immunosorbent assay. Subgroup-specific PCR readily detected ALV-A directly in the contaminated vaccines but not in naive vaccines or cell controls. Our methods are proposed as complementary procedures to the currently required complement fixation for avian leukosis test for detection of ALV in commercial poultry vaccines.

Modeling the Peptide−T Cell Receptor Interaction by the Comparative Molecular Similarity Indices Analysis−Soft Independent Modeling of Class Analogy Technique

A set of 38 epitopes and 183 non-epitopes, which bind to alleles of the HLA-A3 supertype, was subjected to a combination of comparative molecular similarity indices analysis (CoMSIA) and soft independent modeling of class analogy (SIMCA). During the process of T cell recognition, T cell receptors (TCR) interact with the central section of the bound nonamer peptide; thus only positions 4-8 were considered in the study. The derived model distinguished 82% of the epitopes and 73% of the non-epitopes after cross-validation in five groups. The overall preference from the model is for polar amino acids with high electron density and the ability to form hydrogen bonds. These so-called "aggressive" amino acids are flanked by small-sized residues, which enable such residues to protrude from the binding cleft and take an active role in TCR-mediated T cell recognition. Combinations of "aggressive" and "passive" amino acids in the middle part of epitopes constitute a putative TCR binding motif.

Design of natural killer T cell activators: structure and function of a microbial glycosphingolipid bound to mouse CD1d.

Natural killer T (NKT) cells provide an innate-type immune response upon T cell receptor interaction with CD1d-presented antigens. We demonstrate through equilibrium tetramer binding and antigen presentation assays with Valpha14i-positive NKT cell hybridomas that the Sphingomonas glycolipid alpha-galacturonosyl ceramide (GalA-GSL) is a NKT cell agonist that is significantly weaker than alpha-galactosylceramide (alpha-GalCer), the most potent known NKT agonist. For GalA-GSL, a shorter fatty acyl chain, an absence of the 4-OH on the sphingosine tail and a 6'-COOH group on the galactose moiety account for its observed antigenic potency. We further determined the crystal structure of mCD1d in complex with GalA-GSL at 1.8-A resolution. The overall binding mode of GalA-GSL to mCD1d is similar to that of the short-chain alpha-GalCer ligand PBS-25, but its sphinganine chain is more deeply inserted into the F' pocket due to alternate hydrogen-bonding interactions between the sphinganine 3-OH with Asp-80. Subsequently, a slight lateral shift (>1 A) of the galacturonosyl head group is noted at the CD1 surface compared with the galactose of alpha-GalCer. Because the relatively short C(14) fatty acid of GalA-GSL does not fully occupy the A' pocket, a spacer lipid is found that stabilizes this pocket. The lipid spacer was identified by GC/MS as a mixture of saturated and monounsaturated palmitic acid (C(16)). Comparison of available crystal structures of alpha-anomeric glycosphingolipids now sheds light on the structural basis of their differential antigenic potency and has led to the design and synthesis of NKT cell agonists with enhanced cell-based stimulatory activities compared with alpha-GalCer.

Nuclear accumulation of the AT1 receptor in a rat vascular smooth muscle cell line: effects upon signal transduction and cellular proliferation

The objective of the study was to identify the functional outcome of intracellular versus extracellular angiotensin II-AT(1) receptor interactions in vascular cells. Rat vascular smooth muscle cell line A10 was transfected, independently and concurrently, with plasmids encoding fluorescent fusion proteins of rat angiotensin II (pECFP/AII, encodes AII fused downstream of enhanced cyan fluorescent protein) and the rat AT(1a) receptor (pAT(1)R/EYFP, encodes the rat AT(1a) receptor fused upstream of enhanced yellow fluorescent protein). The AII fluorescent fusion protein possesses no secretory signal peptide and deconvolution microscopy established that is maintained within these cells predominantly in the nucleus. AT(1)R/EYFP was absent from the nucleus when expressed exclusively or in untreated cells but accumulated in the nucleus following exogenous AII treatment or when co-expressed with ECFP/AII. Furthermore, expression of ECFP/AII stimulated proliferation of A10 vascular smooth muscle cells (VSMCs) 1.6-fold (P < 0.05). Transfection of a control, pECFP/AII(C) (which encodes a scrambled AII peptide fused to ECFP) had no growth effect. In light of the intracellular growth effects of ECFP/AII, we sought to elucidate the underlying signaling pathways. We found that extracellular AII treatment of A10 cells activated cAMP response element-binding protein (CREB) as determined by one-hybrid assays and immunoblots. Expression of intracellular ECFP/AII similarly activated CREB. However, intracellular and extracellular AII activated CREB through different phosphorylation pathways. Exogenous AII treatment of A10 cells activated p38MAPK and ERK1/2 phosphorylation as determined by Western blot analyses and one-hybrid assays. The p38MAPK inhibitor, SB203580, and the ERK kinase inhibitor, PD98059 each partially inhibited exogenous AII-conferred CREB activation confirming that p38MAPK and ERK1/2 mediate CREB phosphorylation in this system. In contrast, expression of ECFP/AII (intracellular AII) in A10 VSMCs activated p38MAPK but not ERK1/2; inhibition of p38MAPK by SB203580 inhibited intracellular AII-induced CREB phosphorylation. In summary, extracellular AII stimulates at least one pathway common to intracellular AII. This common pathway, in the case of exogenous AII, likely reflects intracellular signaling following internalization of receptor-ligand complex. Extracellular AII also stimulates a unique pathway, apparently reflecting interaction with plasma membrane-associated AT(1)R.

Nonstimulatory peptides contribute to antigen-induced CD8-T cell receptor interaction at the immunological synapse.

It is unclear if the interaction between CD8 and the T cell receptor (TCR)-CD3 complex is constitutive or antigen induced. Here, fluorescence resonance energy transfer microscopy between fluorescent chimeras of CD3zeta and CD8beta showed that this interaction was induced by antigen recognition in the immunological synapse. Nonstimulatory endogenous or exogenous peptides presented simultaneously with antigenic peptides increased the CD8-TCR interaction. This finding indicates that the interaction between the intracellular regions of a TCR-CD3 complex recognizing its cognate peptide-major histocompatibility complex (MHC) antigen, and CD8 (plus the kinase Lck), is enhanced by a noncognate CD8-MHC interaction. Thus, the interaction of CD8 with a nonstimulatory peptide-MHC complex helps mediate T cell recognition of antigen, improving the coreceptor function of CD8.

Group A Streptococcal Surface GAPDH, SDH, Recognizes uPAR/CD87 as its Receptor on the Human Pharyngeal Cell and Mediates Bacterial Adherence to Host Cells

Streptococcal surface dehydrogenase (SDH) is a multifunctional, anchorless protein present on the surface of group A Streptococcus (GAS). It plays a regulatory role in GAS-mediated intracellular signaling events in human pharyngeal cells. Using ligand-binding assays, we have identified an approximately 55 kDa protein as an SDH-specific receptor protein on the surface of Detroit human pharyngeal cells. LC-MS/MS analyses identified this SDH-binding pharyngeal cell-surface-exposed membrane-bound protein as uPAR (urokinase plasminogen activator receptor)/CD87. Ligand-binding assays also revealed that only the N-terminal domain (D1) of uPAR bound to SDH. uPAR-D1 more specifically bound to the C-terminal alpha-helix and two immediate flanking regions of the S-loop of the SDH molecule. Site-directed mutagenesis in GAS resulting in SDH with altered C-terminal ends, and the removal of uPAR from pharyngeal cells by phosphatidylinositol-phopsholipase C treatment decreased GAS ability to adhere to pharyngeal cells. When compared to uninfected Detroit pharyngeal cells, GAS-infected pharyngeal cells showed a transient but a significant increase in the expression of uPAR-specific mRNA, and a prolonged recycling process of uPAR on the cell surface. Together, these results indicate that the specific streptococcal surface protein-pharyngeal cell receptor interaction mediated by SDH and uPAR is modulated during GAS infection of human pharyngeal cells. This interaction significantly contributes to bacterial adherence and thus may play a significant role in GAS pathogenesis by regulating intracellular signaling events in pharyngeal cells.

Intracellular angiotensin II expression alters the distribution of the AT 1 receptor in VSMCs: Effects upon signal transduction and cellular proliferation

The objective of the study was to identify the functional outcome of intracellular versus extracellular angiotensin II:AT1 receptor interactions in vascular cells. Rat vascular smooth muscle cells (line A10) were transfected, independently and concurrently, with plasmids encoding fluorescent fusion proteins of rat angiotensin II (pECFP/AII) and the rat AT1a receptor (pAT1R/EYFP). The AII fusion protein possesses no secretory signal peptide and is maintained within these cells predominantly in the nucleus. AT1R/EYFP is absent from the nucleus when expressed exclusively or in untreated cells. AT1R/EYFP accumulates in the nucleus following exogenous AII treatment or when co-expressed with ECFP/AII. Furthermore, co-expression of ECFP/AII with AT1R/EYFP stimulates proliferation of VSMCs more than 40% (p < 0.001) compared to expression of either protein alone. Co-transfection of a control, pECFP/AIIC (which encodes a scrambled AII peptide fused to ECFP), with pAT1R/EYFP, has no growth effect. In view of the intracellular growth effects of ECFP/AII, we sought to elucidate the underlying signaling pathways. We found that extracellular AII treatment of AT1R/EYFP-expressing VSMCs activates cAMP response element-binding protein (CREB) as determined by one-hybrid assays. Co-expression of intracellular ECFP/AII with AT1R/EYFP similarly activates CREB. However, intracellular and extracellular AII activate CREB through different phosphorylation pathways. Exogenous AII treatment of AT1R/EYFP-expressing VSMCs activates p38MAPK and ERK1/2 phosphorylation as determined by western blot. The p38MAPK inhibitor, SB203580, and the ERK kinase inhibitor, PD98059, each partially inhibit exogenous AII-conferred CREB activation confirming that p38MAPK and ERK1/2 mediate CREB phosphorylation. In contrast, co-expression of ECFP/AII (intracellular AII) and AT1R/EYFP in VSMCs activates p38MAPK but not ERK1/2; inhibition of p38MAPK by SB203580 inhibits intracellular AII-induced CREB phosphorylation. Extracellular AII stimulates at least one pathway common to intracellular AII. This common pathway, in the case of exogenous AII, likely reflects intracellular signaling following internalization of receptor:ligand complex. Extracellular AII also stimulates a unique pathway, reflecting interaction with plasma membrane-associated AT1R.