The CART receptors: background and recent advances

Previous evidence obtained from several behavioral and biochemical studies suggested the existence of multiple CART receptors. However, identification of CART receptor binding has been largely unsuccessful until recently. The first evidence of CART signaling properties came from a study demonstrating that CART 55-102 inhibited voltage-dependent intracellular calcium signaling. More recent studies showed CART-induced dose- and time-dependent activation of extracellular signal-regulated kinase (ERK) 1 and 2 in AtT20 cell line. The activation of ERK was blocked by pertussis toxin but not genisten suggesting the involvement of Gi/o linked cascade in CART's signaling properties in AtT20 cells. Shortly after these findings, the evidence of CART 61-102 specific binding was obtained from the same cell line. This study demonstrated that [(125)I]-CART 61-102 was displaced only by active CART peptide but not by inactive CART fragments or several other unrelated peptides or drugs. The [(125)I]-CART 61-102 binding was saturable and it had a high affinity for a single site in AtT20 cells. The binding was also dependent on time, pH, temperature and protein concentration. The average (+/-S.E.M.) B(max) and K(d) values were 101.4+/-8.8 fmol/mg protein and 21.9+/-8.0 pM, respectively. These data indicate the existence of specific CART receptor binding in AtT20 cells where CART signaling has been demonstrated. The identification of a receptor clone in these cells may help us elucidate CART receptors in other tissues. Because CART is implicated with several physiological functions including feeding, drug reward and stress, identification of a CART receptor would provide a novel target for the development of pharmacological tools and drugs for obesity and other disorders.     

Rat gro/melanoma growth-stimulating activity. Assessment of the structure responsible for chemotactic activity by use of its fragments prepared by proteolysis and chemical synthesis.

Rat gro/melanoma growth-stimulating activity is a dimer composed of two identical subunits. Each subunit consists of 72 amino-acid residues and contains two disulfide bridges. In order to obtain information on the structure responsible for chemotactic activity, various fragments of gro were prepared and tested for their ability to induce chemotaxis. None of the fragments corresponding to residues 1-6, 1-21, 12-31, 36-50 or 52-72 was active as a chemoattractant. Reduced and carboxymethylated gro as well as the tryptic peptide consisting of three peptides, residues 9-21, 28-45 were and 49-61, linked by two disulfide bonds Cys-9-Cys-35 and Cys-11-Cys-51, were inactive. Also, these, peptides did not inhibit the chemotactic activity of gro. Rat gro lacking the N-terminal 6 residues had a reduced activity and the one lacking the C-terminal Lys was as active as intact gro. Therefore, an almost entire portion of the molecule including disulfide cross-links is required for chemotactic activity.     

Amino acid sequence of Streptomyces metallo-proteinase inhibitor from Streptomyces nigrescens TK-23

Streptomyces Metallo-Proteinase Inhibitor (S-MPI) consists of 102 amino acid residues, including one methionine and two disulfide bridges. The complete amino acid sequence of S-MPI, including two disulfide bridges, was determined by sequencing of tryptic and chymotryptic peptides of two fragments obtained by cyanogen bromide cleavage followed by reduction and S-pyridylethylation of the protein. Incubation of the inhibitor with thermolysin slowly cleaved one peptide bond, Cys(64)-Val(65), which might be a reactive site of S-MPI.     

Suitable experimental conditions are required to characterize interferon-alpha2b synthetic peptides.

The binding and antiproliferative activities of synthetic peptides 29-35 and 122-139 of interferon-alpha2b, both of which contain a cysteine residue in their sequences, were studied in the presence or absence of a dissociation medium containing mainly urea, dithiothreitol and 2-mercaptoethanol. Although interferon-alpha2b peptides either did not modify or slightly increased 125I-labelled interferon-alpha2b specific binding to WISH cell-membrane receptors in the absence of dissociation medium, significant binding inhibition was obtained when both peptides were assayed in dissociation medium. Furthermore, also in the presence of dissociating agents, the two fragments inhibited cell growth in a concentration-dependent manner, the 122-139 sequence being more effective than the 29-35 sequence. No additive effect on interferon binding and cell proliferation was observed when both peptides were added simultaneously. Results obtained after submitting peptide 122-139 to gel filtration or PAGE under different experimental conditions showed the presence of dimers and/or noncovalent aggregates arising from intermolecular disulfide bridges or hydrophobic interactions. Thus, our results indicated that peptide effects on 125I-labelled interferon-alpha2b binding and WISH cell proliferation were clearly manifested when the amount of monomeric species increased, showing that suitable experimental conditions should be used to study peptide behavior. The ability of both peptides to effectively trigger an interferon-specific biological action, such as cell growth inhibition, strongly suggested that 29-35 and 122-139 interferon-alpha2b fragments constitute the conformational epitope or mimotope that interacts with the cytokine-specific receptor.     

Epitopic analysis by anti-I-Ak monoclonal antibodies of I-Ak-restricted presentation of lysozyme peptides

Anti-I-A mAb were used as probes of functional epitopes for both the presentation of hen egg lysozyme (HEL) peptides to I-Ak-restricted T cell hybridomas and the direct binding of the HEL (46-61) peptide. When mAb directed to polymorphic regions of I-Ak were used as inhibitors of Ag presentation, several different patterns of inhibition were observed among T cells specific for the same HEL peptide as well as among T cells specific for different fragments of HEL. Although there appears to be a conserved usage of some TCR V beta gene segments among the T cell hybrids specific for the same HEL peptide, no correlation is evident between a single V gene usage and susceptibility to blocking of Ag presentation by a particular anti-I-Ak mAb. Several of the mAb demonstrated T cell "clonotypic blocking" of Ag presentation, whereas others blocked presentation to every T cell hybrid tested, regardless of the peptide specificity. When mAb directed to nonpolymorphic regions of the I-A molecule were tested for their ability to block Ag presentation, little or no inhibition was observed. In addition, Fab' fragments of inhibitory mAb functioned identically to their intact homologous counterparts in their ability to block Ag presentation indicating that "nonspecific" steric hindrance was not playing a major role in the inhibitions observed. When the polymorphic region-directed anti-I-A mAb were tested for their ability to block the direct binding of the lysozyme peptide HEL(46-61) to I-Ak, those mAb that block HEL presentation to all T cell hybrids were found to block the binding of this peptide. However, anti-I-A mAb that demonstrate selective inhibition of T cell hybrid stimulation during Ag presentation, i.e., those directed to polymorphic serologic specificities Ia.15 and Ia.19, do not block the binding of HEL(46-61) to I-Ak. These data indicate that functionally independent epitopes exist on the I-Ak molecule for the binding of antigenic peptides and for interaction with the TCR.     

Protein structural requirements and properties of membrane binding by gamma-carboxyglutamic acid-containing plasma proteins and peptides

The membrane-binding characteristics of a number of modified vitamin K-dependent proteins and peptides showed a general pattern of structural requirements. The amino-terminal peptides from human prothrombin (residues 1-41 and 1-44, 60:40) bovine factor X (residues 1-44), and bovine factor IX (residues 1-42), showed a general requirement for a free amino-terminal group, an intact disulfide, and the tyrosine homologous to Tyr44 of factor X for membrane binding. Consequently, the peptide from factor IX did not bind to membranes. Any of several modifications of the amino terminus, except reaction with trinitrobenzenesulfonic acid, abolished membrane binding by the factor X and prothrombin peptides. Calcium, but not magnesium, protected the amino terminus from chemical modification. The requirement for a free amino terminus was also shown to be true for intact prothrombin fragment 1, factor X, and factor IX. Although aggregation of the peptide-vesicle complexes greatly complicated accurate estimation of equilibrium binding constants, results with the factor X peptide indicated an affinity that was not greatly different from that of the parent protein. The most striking difference shown by the peptides was a requirement for about 10 times as much calcium as the parent proteins. In a manner similar to the parent proteins, the prothrombin and factor X peptides showed a large calcium-dependent quenching of tryptophan fluorescence. This fluorescence quenching in the peptides also required about 10 times the calcium needed by the parent proteins. Thus, the 1-45 region of the vitamin K-dependent proteins contained most of the membrane-binding structure but lacked component(s) needed for high affinity calcium binding. Protein S that was modified by thrombin cleavage at Arg52 and Arg70 showed approximately the same behavior as the amino-terminal 45-residue peptides. That is, it bound to membranes with overall affinity that was similar to native protein S but required high calcium concentrations. These results suggested that the second disulfide loop of protein S (Cys47-Cys72) and prothrombin (Cys48-Cys61) were involved in high affinity calcium binding. Since factor X lacks a homologous disulfide loop, an alternative structure must serve a similar function. A striking property of protein S was dissociation from membranes by high calcium. While this property was shared by all the vitamin K-dependent proteins, protein S showed this most dramatically and supported protein-membrane binding by calcium bridging.     

Complete amino acid sequence and location of the five disulfide bridges in porcine pancreatic alpha-amylase

Porcine pancreatic alpha-amylase (1,4-alpha-D-glucan glucanohydrolase EC 3.2.1.1), a single polypeptide chain, contains nine residues of methionine. Eight different fragments resulting from cleavage of this molecule by cyanogen bromide were characterized. The sequences of six of them have previously been reported. Two missing fragments, CN2 (82 residues) and CN3b1 (76 residues) were purified after breaking of the interpeptidic disulfide bridge and their complete sequence as well as that of the previously purified CN1 peptide (102 residues) are reported here. The location of the three disulfide bridges present in these peptides was determined. Ordering of the carboxymethylated cyanogen bromide fragments was carried out by pulse labeling the amylase chain in vivo. The complete sequence of the porcine pancreatic amylase chain (496 residues) and the location of its five disulfide bridges is presented. Comparison with human and mouse pancreatic and salivary alpha-amylases and with rat pancreatic amylase obtained from the corresponding cDNA nucleotidic sequences shows a high degree of homology between mammalian alpha-amylases.