Membrane-associated and soluble granulocyte/macrophage-colony-stimulating factor receptor alpha subunits are independently regulated in HL-60 cells.
The effects of granulocyte/macrophage-colony-stimulating factor (GM-CSF) are mediated by interaction with its composite receptor (GMR), which consists of a unique alpha subunit (GMR alpha) and a beta subunit (GMR beta) that is common to the receptors for GM-CSF, interleukin 3, and interleukin 5. GMR beta is required for high-affinity binding, cell proliferation, and protein phosphorylation but has no intrinsic GM-CSF-binding activity. GMR alpha in isolation binds to GM-CSF with low affinity and can signal for increased glucose uptake. In addition to the membrane-bound receptor (mGMR alpha), there is a naturally occurring soluble isoform (sGMR alpha) that is released free into the pericellular milieu. Analysis of genomic sequences reveals that the soluble GMR alpha isoform comes about by alternative mRNA splicing. To examine GMR alpha expression, we developed a quantitative reverse transcription-polymerase chain reaction assay based on serial dilutions of in vitro transcribed GMR alpha RNA. This assay provides a strict log-log measure of GMR alpha RNA expression, distinguishes transcripts related to the soluble and membrane-associated isoforms, and quantitatively detects 0.1 fg of GMR alpha-related mRNA. There was little or no GMR alpha expression in two human lymphoid cell lines and in the erythroblastic leukemia cell line K562, but all myeloid cell lines tested expressed both the membrane-associated and soluble isoforms of GMR alpha. Baseline level of expression of both isoforms varied > 20-fold among the myeloid cell lines studied. Differentiation of HL-60 cells to neutrophils with dimethyl sulfoxide led to a 2-fold downregulation of sGMR alpha and a 20-fold upregulation of mGMR alpha. These differentiation-induced transcriptional changes were unrelated to changes in mRNA stability. These findings indicate that sGMR alpha is differentially expressed from mGMR alpha in human hematopoietic cells and that programmed downregulation of sGMR alpha may be important in myeloid maturation.
Heaney
M L
ML
Division of Hematologic Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
Vera
J C
JC
Raines
M A
MA
Golde
D W
DW
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D017931:DNA Primers; D018076:DNA, Complementary; D015972:Gene Expression Regulation, Neoplastic; D015698:Genomic Library; D006801:Humans; D007938:Leukemia; D015473:Leukemia, Promyelocytic, Acute; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis*; D018123:Receptors, Interleukin / Q000096:biosynthesis; D016185:Receptors, Interleukin-3 / Q000096:biosynthesis; D053649:Receptors, Interleukin-5; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D018076:DNA, Complementary; D046911:Macromolecular Substances; D012333:RNA, Messenger; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D018123:Receptors, Interleukin; D016185:Receptors, Interleukin-3; D053649:Receptors, Interleukin-5