Identification of the soluble granulocyte-macrophage colony stimulating factor receptor protein in vivo.
On the basis of the finding of alternatively spliced mRNAs, the alpha-subunit of the receptor for GM-CSF is thought to exist in both a membrane spanning (tmGMRalpha) and a soluble form (solGMRalpha). However, only limited data has been available to support that the solGMRalpha protein product exists in vivo. We hypothesized that hematopoietic cells bearing tmGMRalpha would have the potential to also produce solGMRalpha. To test this hypothesis we examined media conditioned by candidate cells using functional, biochemical, and immunologic means. Three human leukemic cell lines that express tmGMRalpha (HL60, U937, THP1) were shown to secrete GM-CSF binding activity and a solGMRalpha-specific band by Western blot, whereas a tmGMRalpha-negative cell line (K562) did not. By the same analyses, leukapheresis products collected for autologous and allogeneic stem cell transplants and media conditioned by freshly isolated human neutrophils also contained solGMRalpha. The solGMRalpha protein in vivo displayed the same dissociation constant (Kd = 2-5 nmol) as that of recombinant solGMRalpha. A human solGMRalpha ELISA was developed that confirmed the presence of solGMRalpha in supernatant conditioned by the tmGMRalpha-positive leukemic cell lines, hematopoietic progenitor cells, and neutrophils. Furthermore, the ELISA demonstrated a steady state level of solGMRalpha in normal human plasma (36 +/- 17 pmol) and provided data suggesting that plasma solGMRalpha levels can be elevated in acute myeloid leukemias. (Blood. 2000;95:461-469)
Sayani
F
F
Alberta Bone Marrow/Stem Cell Transplant Program and Division of Hematology, Departments of Medicine and Oncology, University of Calgary, Calgary, Alberta, Canada.
Montero-Julian
F A
FA
Ranchin
V
V
Prevost
J M
JM
Flavetta
S
S
Zhu
W
W
Woodman
R C
RC
Brailly
H
H
Brown
C B
CB
D017398:Alternative Splicing; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D018922:HL-60 Cells; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D020014:K562 Cells; D007700:Kinetics; D007937:Leukapheresis; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012044:Regression Analysis; D014162:Transfection; D020298:U937 Cells
D020033:Protein Isoforms; D012333:RNA, Messenger; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor