Design, synthesis, and biological characterization of a peptide-mimetic antagonist for a tethered-ligand receptor

Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G protein-coupled receptors, which are enzymatically cleaved to expose a truncated extracellular N terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease α-thrombin, is expressed in various tissues (e.g., platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. We have discovered a series of potent peptide-mimetic antagonists of PAR-1, exemplified by RWJ-56110. Spatial relationships between important functional groups of the PAR-1 agonist peptide epitope SFLLRN were employed to design and synthesize candidate ligands with appropriate groups attached to a rigid molecular scaffold. Prototype RWJ-53052 was identified and optimized via solid-phase parallel synthesis of chemical libraries. RWJ-56110 emerged as a potent, selective PAR-1 antagonist, devoid of PAR-1 agonist and thrombin inhibitory activity. It binds to PAR-1, interferes with PAR-1 calcium mobilization and cellular function (platelet aggregation; cell proliferation), and has no effect on PAR-2, PAR-3, or PAR-4. By flow cytometry, RWJ-56110 was confirmed as a direct inhibitor of PAR-1 activation and internalization, without affecting N-terminal cleavage. At high concentrations of α-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, albeit not in human platelets; whereas, at high concentrations of SFLLRN-NH2, RWJ-56110 blocked activation responses in both cell types. Thus, thrombin activates human platelets independently of PAR-1, i.e., through PAR-4, which we confirmed by PCR analysis. Selective PAR-1 antagonists, such as RWJ-56110, should serve as useful tools to study PARs and may have therapeutic potential for treating thrombosis and restenosis.

Recognition principle of the TAP transporter disclosed by combinatorial peptide libraries

Transport of peptides across the membrane of the endoplasmic reticulum for assembly with MHC class I molecules is an essential step in antigen presentation to cytotoxic T cells. This task is performed by the major histocompatibility complex-encoded transporter associated with antigen processing (TAP). Using a combinatorial approach we have analyzed the substrate specificity of human TAP at high resolution and in the absence of any given sequence context, revealing the contribution of each peptide residue in stabilizing binding to TAP. Human TAP was found to be highly selective with peptide affinities covering at least three orders of magnitude. Interestingly, the selectivity is not equally distributed over the substrate. Only the N-terminal three positions and the C-terminal residue are critical, whereas effects from other peptide positions are negligible. A major influence from the peptide backbone was uncovered by peptide scans and libraries containing d amino acids. Again, independent of peptide length, critical positions were clustered near the peptide termini. These approaches demonstrate that human TAP is selective, with residues determining the affinity located in distinct regions, and point to the role of the peptide backbone in binding to TAP. This binding mode of TAP has implications in an optimized repertoire selection and in a coevolution with the major histocompatibility complex/T cell receptor complex.

Hepatitis C virus and other Flaviviridae viruses enter cells via low density lipoprotein receptor

Endocytosis of the Flaviviridae viruses, hepatitis C virus, GB virus C/hepatitis G virus, and bovine viral diarrheal virus (BVDV) was shown to be mediated by low density lipoprotein (LDL) receptors on cultured cells by several lines of evidence: by the demonstration that endocytosis of these virus correlated with LDL receptor activity, by complete inhibition of detectable endocytosis by anti-LDL receptor antibody, by inhibition with anti-apolipoprotein E and -apolipoprotein B antibodies, by chemical methods abrogating lipoprotein/LDL receptor interactions, and by inhibition with the endocytosis inhibitor phenylarsine oxide. Confirmatory evidence was provided by the lack of detectable LDL receptor on cells known to be resistant to BVDV infection. Endocytosis via the LDL receptor was shown to be mediated by complexing of the virus to very low density lipoprotein or LDL but not high density lipoprotein. Studies using LDL receptor-deficient cells or a cytolytic BVDV system indicated that the LDL receptor may be the main but not exclusive means of cell entry of these viruses. Studies on other types of viruses indicated that this mechanism may not be exclusive to Flaviviridae but may be used by viruses that associate with lipoprotein in the blood. These findings provide evidence that the family of LDL receptors may serve as viral receptors.

A T cell receptor antagonist peptide induces T cells that mediate bystander suppression and prevent autoimmune encephalomyelitis induced with multiple myelin antigens

Experimental autoimmune encephalomyelitis (EAE) induced with myelin proteolipid protein (PLP) residues 139–151 (HSLGKWLGHPDKF) can be prevented by treatment with a T cell receptor (TCR) antagonist peptide (L144/R147) generated by substituting at the two principal TCR contact residues in the encephalitogenic peptide. The TCR antagonist peptide blocks activation of encephalitogenic Th1 helper cells in vitro, but the mechanisms by which the antagonist peptide blocks EAE in vivo are not clear. Immunization with L144/R147 did not inhibit generation of PLP-(139–151)-specific T cells in vivo. Furthermore, preimmunization with L144/R147 protected mice from EAE induced with the encephalitogenic peptides PLP-(178–191) and myelin oligodendrocyte protein (MOG) residues 92–106 and with mouse myelin basic protein (MBP). These data suggest that the L144/R147 peptide does not act as an antagonist in vivo but mediates bystander suppression, probably by the generation of regulatory T cells. To confirm this we generated T cell lines and clones from animals immunized with PLP-(139–151) plus L144/R147. T cells specific for L144/R147 peptide were crossreactive with the native PLP-(139–151) peptide, produced Th2/Th0 cytokines, and suppressed EAE upon adoptive transfer. These studies demonstrate that TCR antagonist peptides may have multiple biological effects in vivo. One of the principal mechanisms by which these peptides inhibit autoimmunity is by the induction of regulatory T cells, leading to bystander suppression of EAE. These results have important implications for the treatment of autoimmune diseases where there are autopathogenic responses to multiple antigens in the target organ.

Tumor necrosis factor (TNF)-mediated kinase cascades: Bifurcation of Nuclear Factor-κB and c-jun N-terminal kinase (JNK/SAPK) pathways at TNF receptor-associated factor 2

TNF-induced activation of the transcription factor NF-κB and the c-jun N-terminal kinase (JNK/SAPK) requires TNF receptor-associated factor 2 (TRAF2). The NF-κB-inducing kinase (NIK) associates with TRAF2 and mediates TNF activation of NF-κB. Herein we show that NIK interacts with additional members of the TRAF family and that this interaction requires the conserved “WKI” motif within the TRAF domain. We also investigated the role of NIK in JNK activation by TNF. Whereas overexpression of NIK potently induced NF-κB activation, it failed to stimulate JNK activation. A kinase-inactive mutant of NIK was a dominant negative inhibitor of NF-κB activation but did not suppress TNF- or TRAF2-induced JNK activation. Thus, TRAF2 is the bifurcation point of two kinase cascades leading to activation of NF-κB and JNK, respectively.

Functional cleavage of the common cytokine receptor γ chain (γc) by calpain

The small subunit of calpain, a calcium-dependent cysteine protease, was found to interact with the cytoplasmic domain of the common cytokine receptor γ chain (γc) in a yeast two-hybrid interaction trap assay. This interaction was functional as demonstrated by the ability of calpain to cleave in vitro-translated wild-type γc, but not γc containing a mutation in the PEST (proline, glutamate, serine, and threonine) sequence in its cytoplasmic domain, as well as by the ability of endogenous calpain to mediate cleavage of γc in a calcium-dependent fashion. In T cell receptor-stimulated murine thymocytes, calpain inhibitors decreased cleavage of γc. Moreover, in single positive CD4+ thymocytes, not only did a calpain inhibitor augment CD3-induced proliferation, but antibodies to γc blocked this effect. Finally, treatment of cells with ionomycin could inhibit interleukin 2-induced STAT protein activation, but this inhibition could be reversed by calpain inhibitors. Together, these data suggest that calpain-mediated cleavage of γc represents a mechanism by which γc-dependent signaling can be controlled.

Inter- and intraspecies polymorphisms in the cholecystokinin-B/gastrin receptor alter drug efficacy

The brain cholecystokinin-B/gastrin receptor (CCK-BR) is a major target for drug development because of its postulated role in modulating anxiety, memory, and the perception of pain. Drug discovery efforts have resulted in the identification of small synthetic molecules that can selectively activate this receptor subtype. These drugs include the peptide-derived compound PD135,158 as well as the nonpeptide benzodiazepine-based ligand, L-740,093 (S enantiomer). We now report that the maximal level of receptor-mediated second messenger signaling that can be achieved by these compounds (drug efficacy) markedly differs among species homologs of the CCK-BR. Further analysis reveals that the observed differences in drug efficacy are in large part explained by single or double aliphatic amino acid substitutions between respective species homologs. This interspecies variability in ligand efficacy introduces the possibility of species differences in receptor-mediated function, an important consideration when selecting animal models for preclinical drug testing. The finding that even single amino acid substitutions can significantly affect drug efficacy prompted us to examine ligand-induced signaling by a known naturally occurring human CCK-BR variant (glutamic acid replaced by lysine in position 288; 288E → K). When examined using the 288E → K receptor, the efficacies of both PD135,158 and L-740,093 (S) were markedly increased compared with values obtained with the wild-type human protein. These observations suggest that functional variability resulting from human receptor polymorphisms may contribute to interindividual differences in drug effects.

A peptide derived from an extracellular domain selectively inhibits receptor internalization: Target sequences on insulin and insulin-like growth factor 1 receptors

Certain peptides derived from the α1 domain of the major histocompatibility class I antigen complex (MHC-I) inhibit receptor internalization, increasing the steady-state number of active receptors on the cell surface and thereby enhancing the sensitivity to hormones and other agonists. These peptides self-assemble, and they also bind to MHC-I at the same site from which they are derived, suggesting that they could bind to receptor sites with significant sequence similarity. Receptors affected by MHC-I peptides do, indeed, have such sequence similarity, as illustrated here by insulin receptor (IR) and insulin-like growth factor-1 receptor. A synthetic peptide with sequence identical to a certain extracellular receptor domain binds to that receptor in a ligand-dependent manner and inhibits receptor internalization. Moreover, each such peptide is selective for its cognate receptor. An antibody to the IR peptide not only binds to IR and competes with the peptide but also inhibits insulin-dependent internalization of IR. These observations, and binding studies with deletion mutants of IR, indicate that the sequence QILKELEESSF encoded by exon 10 plays a key role in IR internalization. Our results illustrate a principle for identifying receptor-specific sites of importance for receptor internalization, and for enhancing sensitivity to hormones and other agonists.

The primary fibrin polymerization pocket: Three-dimensional structure of a 30-kDa C-terminal γ chain fragment complexed with the peptide Gly-Pro-Arg-Pro

After vascular injury, a cascade of serine protease activations leads to the conversion of the soluble fibrinogen molecule into fibrin. The fibrin monomers then polymerize spontaneously and noncovalently to form a fibrin gel. The primary interaction of this polymerization reaction is between the newly exposed N-terminal Gly-Pro-Arg sequence of the α chain of one fibrin molecule and the C-terminal region of a γ chain of an adjacent fibrin(ogen) molecule. In this report, the polymerization pocket has been identified by determining the crystal structure of a 30-kDa C-terminal fragment of the fibrin(ogen) γ chain complexed with the peptide Gly-Pro-Arg-Pro. This peptide mimics the N terminus of the α chain of fibrin. The conformational change in the protein upon binding the peptide is subtle, with electrostatic interactions primarily mediating the association. This is consistent with biophysical experiments carried out over the last 50 years on this fundamental polymerization reaction.

Homeostatic expansion and phenotypic conversion of naïve T cells in response to self peptide/MHC ligands

Recent data suggest that survival of resting, naïve T cells requires an interaction with self MHC molecules. From analysis of the class I MHC-restricted T cell receptor transgenic strain OT-I, we report a different response. Rather than merely surviving, these T cells proliferated slowly after transfer into T-depleted syngeneic hosts. This expansion required both T cell “space” and expression of normal levels of self class I MHC molecules. Furthermore, we demonstrate that during homeostatic expansion in a suitable environment, naïve phenotype (CD44low) OT-I T cells converted to memory phenotype (CD44med/high), despite the absence of foreign antigenic stimulation. On the other hand, cells undergoing homeostatic expansion did not acquire cytolytic effector function. The significance of these data for reactivity of T cells with self peptide/MHC ligands and the implications for normal and abnormal T cell homeostasis are discussed.

Molecular basis for the recognition of two structurally different major histocompatibility complex/peptide complexes by a single T-cell receptor

2C is a typical alloreactive cytotoxic T lymphocyte clone that recognizes two different ligands. These ligands are adducts of the allo-major histocompatibility complex (MHC) molecule H-2Ld and an endogenous octapeptide, and of the self-MHC molecule H-2Kb and another peptide. MHC-binding and T-cell assays with synthetic peptides in combination with molecular modeling studies were employed to analyze the structural basis for this crossreactivity. The molecular surfaces of the two complexes differ greatly in densities and distributions of positive and negative charges. However, modifications of the peptides that increase similarity decrease the capacities of the resulting MHC peptide complexes to induce T-cell responses. Moreover, the roles of the peptides in ligand recognition are different for self- and allo-MHC-restricted T-cell responses. The self-MHC-restricted T-cell responses were finely tuned to recognition of the peptide. The allo-MHC-restricted responses, on the other hand, largely ignore modifications of the peptide. The results strongly suggest that adaptation of the T-cell receptor to the different ligand structures, rather than molecular mimicry by the ligands, is the basis for the crossreactivity of 2C. This conclusion has important implications for T-cell immunology and for the understanding of immunological disorders.

Analysis of a peptide hormone–receptor interaction in the yeast two-hybrid system

Interaction between a peptide hormone and extracellular domains of its receptor is a crucial step for initiation of hormone action. We have developed a modification of the yeast two-hybrid system to study this interaction and have used it to characterize the interaction of insulin-like growth factor 1 (IGF-1) with its receptor by using GAL4 transcriptional regulation with a β-galactosidase assay as readout. In this system, IGF-1 and proIGF-1 bound to the cysteine-rich domain, extracellular domain, or entire IGF-1 proreceptor. This interaction was specific. Thus, proinsulin showed no significant interaction with the IGF-1 receptor, while a chimeric proinsulin containing the C-peptide of IGF-1 had an intermediate interaction, consistent with its affinity for the IGF-1 receptor. Over 2000 IGF-1 mutants were generated by PCR and screened for interaction with the color assay. About 40% showed a strong interaction, 20% showed an intermediate interaction, and 40% give little or no signal. Of 50 mutants that were sequenced, several (Leu-5 → His, Glu-9 → Val, Arg-37 → Gly, and Met-59 → Leu) appeared to enhance receptor association, others resulted in weaker receptor interaction (Tyr-31 → Phe and Ile-43 → Phe), and two gave no detectable signal (Leu-14 → Arg and Glu-46 → Ala). Using PCR-based mutagenesis with proinsulin, we also identified a gain of function mutant (proinsulin Leu-17 → Pro) that allowed for a strong IGF-1–receptor interaction. These data demonstrate that the specificity of the interaction between a hormone and its receptor can be characterized with high efficiency in the two-hybrid system and that novel hormone analogues may be found by this method.

Molecular uncoupling of C-C chemokine receptor 5-induced chemotaxis and signal transduction from HIV-1 coreceptor activity

The C-C chemokine receptor 5 (CCR5) plays a crucial role in facilitating the entry of macrophage-tropic strains of the HIV-1 into cells, but the mechanism of this phenomenon is completely unknown. To explore the role of CCR5-derived signal transduction in viral entry, we introduced mutations into two cytoplasmic domains of CCR5 involved in receptor-mediated function. Truncation of the terminal carboxyl-tail to eight amino acids or mutation of the highly conserved aspartate-arginine-tyrosine, or DRY, sequence in the second cytoplasmic loop of CCR5 effectively blocked chemokine-dependent activation of classic second messengers, intracellular calcium fluxes, and the cellular response of chemotaxis. In contrast, none of the mutations altered the ability of CCR5 to act as an HIV-1 coreceptor. We conclude that the initiation of signal transduction, the prototypic function of G protein coupled receptors, is not required for CCR5 to act as a coreceptor for HIV-1 entry into cells.

Protein kinase C potentiation of N-methyl-d-aspartate receptor activity is not mediated by phosphorylation of N-methyl-d-aspartate receptor subunits

N-methyl-d-aspartate receptors (NMDARs) are Ca2+-permeable glutamate-gated ion channels whose physiological properties in neurons are modulated by protein kinase C (PKC). The present study was undertaken to determine the role in PKC-induced potentiation of the NR1 and NR2A C-terminal tails, which serve as targets of PKC phosphorylation [Tingley, W. G., Ehlers, M. D., Kameyama, K., Doherty, C., Ptak, J. B., Riley, C. T. & Huganir, R. L. (1997) J. Biol. Chem. 272, 5157–5166]. Serine residue 890 in the C1 cassette is a primary target of PKC phosphorylation and a critical residue in receptor clustering at the membrane. We report herein that the presence of the C1 cassette reduces PKC potentiation and that mutation of Ser-890 significantly restores PKC potentiation. Splicing out or deletion of other C-terminal cassettes singly or in combination had little or no effect on PKC potentiation. Moreover, experiments involving truncation mutants reveal the unexpected finding that NMDARs assembled from subunits lacking all known sites of PKC phosphorylation can show PKC potentiation. These results indicate that PKC-induced potentiation of NMDAR activity does not occur by direct phosphorylation of the receptor protein but rather of associated targeting, anchoring, or signaling protein(s). PKC potentiation of NMDAR function is likely to be an important mode of NMDAR regulation in vivo and may play a role in NMDA-dependent long-term potentiation.

A dileucine motif in the C terminus of the β2-adrenergic receptor is involved in receptor internalization

The cytoplasmic C terminus of the β2-adrenergic receptor and many other G protein-coupled receptors contains a dileucine sequence that has been implicated in endosome/lysosome targeting of diverse proteins. In the present study, we provide evidence for an essential role of this motif in the agonist-induced internalization of the β2-adrenergic receptor. Mutation of Leu-339 and/or Leu-340 to Ala caused little changes in surface expression, ligand binding, G protein coupling, and signaling to adenylyl cyclase, when these receptors were transiently or stably expressed in CHO or HEK-293 cells. However, agonist-induced receptor internalization was markedly impaired in the L339,340A double mutant and reduced in the two single mutants. This impairment in receptor internalization was seen by using various approaches to determine internalization: binding of hydrophobic vs. hydrophilic ligands, loss of surface β2-adrenergic receptor immunoreactivity, and immunofluorescence microscopy. The selective effects of these mutations suggest that the C-terminal dileucine motif is involved in agonist-induced internalization of the β2-adrenergic receptor.