Reversible protein phosphorylation is a key regulatory process in all living cells. Deregulation of modification control mechanisms, especially in the case of tyrosine, may lead to malignant transformation and disease. Phosphotyrosine (p-Tyr) accounts for only 0.05% of the total cellular phospho-amino acid content, yet plays an unusually prominent role in eukaryotic signaling, development, and growth. Tracking temporal and positional p-Tyr changes across the cellular proteome, i.e. tyrosine phosphoproteomics, is therefore tremendously valuable. Here, we describe and evaluate a prototype antibody (Ab) microarray platform to monitor changes in protein Tyr phosphorylation. Availability permitting, a virtually unlimited number of Abs, each recognizing a specific cellular protein, may be arrayed on a chip, incubated with total cell or tissue extracts or with biological fluids, and then probed with a fluorescently labeled p-Tyr-specific monoclonal Ab, PY-KD1, specifically generated for this assay as part of the current study. The optimized protocol allowed detection of changes in the Tyr phosphorylation state of selected proteins using submicrogram to low nanogram of total protein extract, amounts that may conceivably be obtained from a thousand to a hundred thousand cells, or less, depending on the cell or tissue type. The assay platform was evaluated by assessing changes in a rationally selected subset of the Tyr phosphoproteome of BcrAbl-expressing cells treated with a specific inhibitor, Gleevec, and of epidermal growth factor (EGF)-treated HeLa cells. The results, ratiometric rather than strictly quantitative in nature, conformed with previous identifications of several Bcr-Abl and EGF receptor targets, and associated proteins, as detected by exhaustive mass spectrometric analyses. The Ab microarray method described here offers advantages of low sample and reagent consumption, scalability, detection multiplexing, and potential compatibility with microfluidic devices and automation. The system may hold particular promise for dissecting signaling pathways, molecular classification of tumors, and profiling of novel target-cancer drugs.
Steric and electrostatic requirements at position 6 of [Nle(10)]NKA(4-10), a full agonist of NK-2 receptors, for molecular recognition by the receptor were studied. Two series of peptide analogues, (a) p-substituted analogues, [p-X-Phe(6), Nle(10)]NKA(4-10), where X = F, Cl, Br, I, NH(2), NO(2), and (b) [D-Phe(6),Nle(10)]NKA(4-10), [Trp(6),Nle(10)]NKA(4-10), and [Chex-Ala(6),Nle(10)]NKA(4-10), were synthesized, and their biological activity was examined. Competition binding experiments with [(3)H]NKA were performed using cloned human NK-2 receptors expressed in CHO cells. Antagonistic and agonistic properties of the analogues were studied using an in vitro functional assay with hamster tracheal rings. The rank order of potency of agonists was [Nle(10)]NKA(4-10) approximately [p-F-Phe(6),Nle(10)]NKA(4-10) > [p-NH(2)-Phe(6),Nle(10)]NKA(4-10) > [p-Cl-Phe(6),Nle(10)]NKA(4-10) > [p-NO(2)-Phe(6),Nle(10)]NKA(4-10) > [Trp(6),Nle(10)]NKA(4-10). Size and planarity of the aromatic side chain were crucially important for the biological activity, whereas electron-donating and electron-withdrawing properties of the para-substituent were less important. The results favor the hypothesis that weakly polar pi-pi interactions exist between the aromatic group and the receptor.
The importance of the C-terminal Phe of gastrin and structural requirements at position 17 for binding to the human CCK2 receptor were assessed using analogs of [Leu15]G(11-17). The following peptides were synthesized, Ac[Leu15]G(11-17), Ac[Leu15]G(11-16)NH2, [Leu15]G(11-17), [Leu15,Ala17]G(11-17), [Leu15,Abu17]G(11-17), [Leu15,Val17]G(11-17), [Leu15,Leu17]G(11-17), [Leu15,Cha17]G(11-17), [Leu15,Trp17]G(11-17), [Leu15,Tic17]G(11-17), [Leu15, d-Phe17]G(11-17) and [Leu15,p-X-Phe17]G(11-17), where X = F, Cl, Br, I, OH, CH3, NH2 and NO2. Competition binding experiments with [3H]CCK-8 were performed using human CCK2 receptors stably expressed in CHO cells. Phe17 was shown to be important for binding. A hydrophobic side-chain larger than Leu is required at position 17 but aromaticity does not appear to be essential. Constraint of the aromatic side-chain either in the g+ or g- conformation, as in the case of Tic, results in a significant decrease in affinity. In addition, the peptide conformation induced by incorporation of d-Phe decreases binding. The size and electron withdrawing/donating properties of the para substituent are not important for interaction with the receptor. The current study shows that the use of des-Phe analogs of gastrin is not a viable strategy for development of antagonists for the human CCK2 receptor.
The melanocyte-inhibiting tripeptide (MTP) pyroGlu-Phe-GlyNH2 is present in tissue cultures of non-transformed melanocytes and melanoma cells and influences melanocyte growth in vitro. The objective of the present study was to investigate a possible effect of MTP on the in vivo growth of B16A2, a monoclonal experimental melanoma. The B16A2 clone was established by the limited dilution technique. It has a reduced DNA content and displays slower growth both in vivo and in vitro compared to the parent cell line (B16). B16A2 cells were injected subcutaneously into hairless mice at four sites (300 000 cells in 0.25 ml buffer/site). MTP was given by i.p. injection 3 times a week at two concentrations (1 pmol and 1 nmol/animal). The control animals received the equal volume of solvent. The animals were sacrificed 1 and 2 weeks after tumour transplantation, and all tumours were weighed. One week after transplantation, the animals who received 1 pmol MTP had fewer tumours and a reduced tumour load. Two weeks after the transplantation, the differences between control and treated animals were no longer observed. The results indicate that MTP temporarily delays in vivo tumour growth.
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