Purification, receptor binding analysis, and biological characterization of human melanoma growth stimulating activity (MGSA). Evidence for a novel MGSA receptor.
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.
Horuk
R
R
Department of Protein Chemistry, Genentech, Inc., South San Francisco, California 94080.
Yansura
D G
DG
Reilly
D
D
Spencer
S
S
Bourell
J
J
Henzel
W
W
Rice
G
G
Unemori
E
E
D000595:Amino Acid Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006133:Growth Substances / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D016209:Interleukin-8 / Q000494:pharmacology; D007700:Kinetics; D013058:Mass Spectrometry; D008545:Melanoma; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D010957:Plasmids; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018121:Receptors, Cytokine; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014407:Tumor Cells, Cultured
C475829:CXCL1 protein, human; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D016209:Interleukin-8; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D018121:Receptors, Cytokine; D011994:Recombinant Proteins; C078862:melanoma growth stimulating activity receptor; D002118:Calcium