Purification, receptor binding analysis, and biological characterization of human melanoma growth stimulating activity (MGSA). Evidence for a novel MGSA receptor
| Autor: | Horuk R, Dg, Yansura, Reilly D, Spencer S, Bourell J, William Henzel, Rice G, Unemori E |
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| Rok vydání: | 1993 |
| Předmět: |
Chemokine CXCL1
Blotting Western Receptors Cell Surface Mass Spectrometry Escherichia coli Tumor Cells Cultured Humans Amino Acid Sequence Cloning Molecular Receptors Cytokine Growth Substances Melanoma Chromatography High Pressure Liquid Binding Sites Chemotactic Factors Interleukin-8 Chromatography Ion Exchange Peptide Fragments Recombinant Proteins Neoplasm Proteins Molecular Weight Kinetics Intercellular Signaling Peptides and Proteins Calcium Electrophoresis Polyacrylamide Gel Chemokines CXC Cell Division Plasmids |
| Zdroj: | Europe PubMed Central |
| ISSN: | 0021-9258 |
| Popis: | Human melanoma growth stimulating activity (MGSA) is a mitogenic factor first identified in the conditioned media of human melanoma cells. Structurally, MGSA belongs to a superfamily of proteins that includes interleukin-8 (IL-8) and platelet factor 4. These proteins are involved in inflammatory processes, and an understanding of their mechanism of action should provide insight into their pathophysiology. In this study, we report the high level expression of recombinant human MGSA in Escherichia coli. The structure was confirmed by mass spectrometry and NH2-terminal amino acid sequencing. Receptor binding studies were carried out in a human melanoma cell line, Hs294T, and in U937 cells. Direct binding experiments with 125I-MGSA in Hs294T cells have allowed us to identify a novel MGSA receptor in these cells, with a KD of 3.9-4.25 nM and approximately 52,960-67,758 binding sites/cell. These MGSA-binding sites were specific and could not be displaced by unlabeled IL-8. The MGSA receptor in these cells is biologically active, and the addition of ligand induces cellular proliferation in a dose-dependent manner. In U937 cells, unlabeled IL-8 and MGSA were able to completely displace radiolabeled IL-8. Scatchard analysis of the displacement binding data was consistent with binding to a single class of binding sites, and the calculated KD values were 2.4 +/- 0.6 nM for IL-8 and 3.2 +/- 0.80 nM for MGSA. Treatment of U937 cells with IL-8 or MGSA produced a rapid increase in Ca2+ flux; however, subsequent incubation with either ligand failed to produce any further Ca2+ flux. The IL-8 receptor in U937 cells was covalently labeled with 125I-IL-8 to reveal a protein with a molecular mass of 69 kDa. |
| Databáze: | OpenAIRE |
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