Functional proteomics of signal transduction by membrane receptors.

Functional proteomic methods have been developed and applied to the investigation of signal transduction systems involving platelet-derived growth factor (PDGF), endothelin and bradykinin receptors. Mouse fibroblast cells have been stimulated with PDGF or endothelin. Phosphorylation/dephosphorylation of several hundred proteins has been followed as a function of time following stimulation using 2-D gel electrophoresis and anti-phosphotyrosine or anti-phosphoserine antibodies. Up to 100 of these proteins showed strong changes in phosphorylation with minutes of receptor stimulation. Identification of some of these proteins by mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and by partial peptide sequencing with ion trap electrospray mass spectrometry has identified proteins which were previously known to be associated with PDGF signaling, proteins which have been shown to be involved in other signaling pathways, but not PDGF and proteins not previously associated with signal transduction. Parallel to these studies, new methods for rapid, single-step isolation of peptide receptors using a peptide coupled to a (dA)30 oligonucleotide have been developed and applied to mass spectrometric studies of post-translational modifications of the endothelin B and bradykinin B2 receptors under in vivo conditions. Both receptors have been shown to undergo extensive phosphorylation as well as palmitoylation. The patterns of post-translational modifications are more complex than previously recognized and provide new indications of possible roles for these modifications in the regulation and response of these receptors.

Functional proteomics analysis of signal transduction pathways of the platelet-derived growth factor beta receptor.

We report efficient methods for using functional proteomics to study signal transduction pathways in mouse fibroblasts following stimulation with PDGF. After stimulation, complete cellular proteins were separated using two-dimensional electrophoresis and phosphorylated proteins were detected with anti-phosphotyrosine and anti-phosphoserine antibodies. About 260 and 300 phosphorylated proteins were detected with the anti-phosphotyrosine and anti-phosphoserine antibodies, respectively, at least 100 of which showed prominent changes in phosphorylation as a function of time after stimulation. Proteins showing major time-dependent changes in phosphorylation were subjected to in-gel digestion with trypsin and identified by mass spectroscopy using MALDI-TOF mass fingerprinting and ESI peptide sequencing. We have observed phosphorylated proteins known to be part of the PDGF signal transduction pathway such as ERK 1, serine/threonine protein kinase akt and protein tyrosine phosphatase syp, proteins such as proto-oncogene tyrosine kinase fgr previously known to participate in other signal transduction pathways, and some proteins such as plexin-like protein with no previously known function in signal transduction. Information about the phosphorylation site was obtained for proto-oncogene tyrosine kinase fgr and for cardiac alpha-actin. The methods used here have proven to be suitable for the identification of time-dependent changes in large numbers of proteins involved in signal transduction pathways.

Post-translational modifications of endothelin receptor B from bovine lungs analyzed by mass spectrometry.

A new mild experimental approach for isolation of peptide membrane receptors and subsequent analysis of post-translational modifications is described. Endothelin receptors A and B were isolated on oligo(dT)-cellulose using N-(epsilon-maleimidocaproyloxy)succinimide endothelin coupled to a protected (dA)-30-mer. This allowed a one-step isolation of the receptor from oligo(dT)-cellulose via variation solely of salt concentration. The identity of the receptor was confirmed by direct amino acid sequencing of electroblotted samples or by using antibodies against ETA and ETB receptors. The method used here is very fast, requires only very mild elution conditions and, for the first time, gave both ETA and ETB receptors concurrently in very good yield. Following enzymatic in-gel digestion, MALDI, and electrospray ion trap mass spectrometric analysis of the isolated endothelin B receptor showed phosphorylation at Ser-304, -418, -438, -439, -440, and -441. Further phosphorylation at either Ser-434 or -435 was observed. The endothelin B receptor is also palmitoylated at Cys residues 402 and 404. Phosphorylation of Ser304 may play a role in Hirschsprung's disease.

Labelling of breast carcinoma, thyroid carcinoma and melanoma with manno- and galacto-specific lectins from marine invertebrates.

Three marine invertebrate FITC-labelled lectins, CNL, GCL, and GSL, isolated respectively, from the sponges Chondrilla nucula, Geodia cydonium, and the hexacoral Gerardia savaglia, were used as potential diagnostic tools for different breast tumors. The lectins vary in their carbohydrate binding properties: GSL is D-mannose specific, GCL and CNL D-galactose specific. GSL labels most investigated types of malignant tissues distinctively, while the results with CNL and GCL are less consistent. The well known D-mannose specific lectin, concanavalin A, also binds to tumor tissues, but with much lower intensity than GSL.

Inhibition of human immunodeficiency virus-1 infection by human conglutinin-like protein: in vitro studies.

The lectin-like protein analogous to bovine conglutinin was purified from human serum. The carbohydrate-binding ability of conglutinin-like protein was inhibited by D-mannose, N-acetylglucosamine and L-fucose as well as by mannan-containing oligosaccharides. By applying a lectin-based ELISA system it was demonstrated that conglutinin-like protein binds to human immunodeficiency virus-1 (HIV-1) glycoprotein 120 (gp120) via its carbohydrate binding site. In vitro experiments with T-lymphoblastoid CEM cells revealed that conglutinin-like protein abolishes infection by HIV-1; a 50% cytoprotective concentration of 23.9 micrograms/ml was measured. These findings demonstrate that human conglutinin-like protein binds to HIV-gp120 and inhibits, under the described in vitro conditions, CEM cell infection.

The lectin from the algae Udotea petiolata: isolation, characterization and sugar binding properties.

In the present work, we describe a novel lectin which is specific for poly-N-acetyllactosamine sequences on complex N- and O-linked carbohydrate chains. This lectin was extracted and purified from the algae Udotea petiolata. The purified lectin is a monomer with a molecular mass of 65,000 and an isoelectric point of 5.6. It agglutinates normal, neuraminidase and protease-treated erythrocytes from humans irrespectively of the blood group (A, B and O) and animal erythrocytes. The Udotea lectin displays a strong mitogenic effect on human lymphocytes, especially T-cells. This lectin binds to the human serum plasma protein 8S alpha 3-glycoprotein with high affinity (ID50 0.02 microM); other species of human serum glycoproteins exhibiting a similar preponderance of complex type N-glycosylation showed also high binding capacities in the order 9.5 S alpha 1-glycoprotein greater than alpha 2-macroglobulin = beta 2 glycoprotein = immunoglobulin A greater than asialofetuin greater than alpha 1-acid glycoprotein and mucin glycopeptide (from amnion fluid). Monosaccharides and disaccharides tested do not bind to the lectin. This novel lectin will be useful for identification of N- and O-linked glycans rich in poly-N-acetyllactosamine.