A 25 kDa polypeptide is the ligand for p185neu and is secreted by activated macrophages.
Medium conditioned by mouse peritoneal macrophages, activated by muramyl dipeptide (MDP), was used as a possible source of p185neu-specific ligand. MDP-activated macrophage-conditioned medium (MDP-CM) was shown to induce p185neu down-regulation in NEU-expressing NIH3T3 cells in a dose-dependent and temperature-sensitive manner. To exclude the possibility of an indirect action of proteins/metabolites present in MDP-CM on p185neu turnover, a ligand-trapping approach was used. Secreted NEU protein possessing only the extracellular domain but lacking transmembrane and protein kinase domains was expressed in HeLa cells and then purified from conditioned medium, using affinity chromatography on WGA-Sepharose. Co-incubation of the truncated, soluble NEU protein preparation with MDP-CM abolished MDP-CM-induced p185neu down-regulation and reduced self-phosphorylation. It is concluded that a putative p185neu-specific ligand is produced by macrophages activated by MDP. Using MDP-CM, the presence of a 25 kDa polypeptide distinct from EGF, PDGF, FGF, IGF, TGF-alpha and TGF-beta and TNF-alpha, could be demonstrated by decorating a Western blot with soluble NEU and anti-NEU antibodies. Thus, a 25 kDa (non-reduced) p185neu ligand has been described.
Department of Leukemiagenesis, Academy of Sciences Ukrainian SSR, Kiev.
A
A
Tarakhovsky
T
T
Zaichuk
V
V
Prassolov
Z A
ZA
Butenko
D016475:3T3 Cells; D000119:Acetylmuramyl-Alanyl-Isoglutamine / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D004261:DNA Replication; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D008262:Macrophage Activation; D008264:Macrophages / Q000187:drug effects / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D011519:Proto-Oncogenes; D018719:Erb-b2 Receptor Tyrosine Kinases; D015183:Restriction Mapping; D014162:Transfection
D000119:Acetylmuramyl-Alanyl-Isoglutamine; D008024:Ligands; D008565:Membrane Proteins; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases