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Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells
Published on: October 2, 2010
Transforming activities of human CSF-1 receptors with different point mutations at codon 301 in their extracellular
M F Roussel1, J R Downing, C J Sherr
1Howard Hughes Medical Institute, St. Jude Children's Research Hospital, Memphis, Tennessee 38105.
Abstract:
Replacement of leucine 301 in the human colony stimulating factor 1 receptor (CSF-1R) by serine, threonine, glutamic acid, or proline induced ligand-independent transforming activity in mouse NIH3T3 cells, whereas substitution by phenylalanine, methionine, cysteine, or lysine did not. Serine, glutamic acid, and proline mutations were more potent than threonine in inducing cell transformation. The growth of cells transformed by CSF-1R [S301] and [T301] was further stimulated by human recombinant CSF-1, but cells expressing CSF-1R [E301] responded poorly to the growth factor. The transforming efficiency of mutant receptors was also enhanced by the presence of a phenylalanine for tyrosine mutation at codon 969 near the receptor carboxylterminus. Like the v-fms oncogene product, receptors containing S301 or E301 mutations were partially inhibited in their intracellular transport to the plasma membrane, whereas non-transforming variants were transported normally. However, CSF-1R [T301] was processed as efficiently as the wild-type glycoprotein, indicating that the properties of altered transport and cell transformation could be at least partially dissociated. Expression of CSF-1 receptors bearing activating mutations led to increased phosphorylation of cellular substrates on tyrosine, suggesting that cell transformation resulted from constitutive receptor kinase activity. We conclude that only particular mutations at codon 301 mimic an effect of ligand on CSF-1R so as to constitutively activate its growth promoting activity.
Insights
Specific mutations at codon 301 in the colony-stimulating factor 1 receptor (CSF-1R) activate its growth-promoting activity without a ligand. These findings reveal how CSF-1R mutations drive cell transformation and cancer. Keywords: CSF-1R, cell transformation, cancer.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- The colony-stimulating factor 1 receptor (CSF-1R) is a key regulator of cell proliferation and survival.
- Dysregulation of CSF-1R signaling is implicated in various cancers.
- Understanding the mechanisms of CSF-1R activation is crucial for developing targeted therapies.
Purpose of the Study:
- To investigate the role of specific amino acid substitutions at codon 301 of CSF-1R in inducing ligand-independent transforming activity.
- To determine the impact of these mutations on receptor function, cell growth, and intracellular transport.
- To elucidate the signaling pathways involved in CSF-1R-mediated cell transformation.
Main Methods:
- Site-directed mutagenesis of the human CSF-1R gene to introduce specific amino acid substitutions at codon 301.
- Transfection of NIH3T3 cells with wild-type and mutant CSF-1R constructs.
- Assays for cell transformation, cell growth stimulation by CSF-1, and receptor intracellular transport.
- Analysis of tyrosine phosphorylation of cellular substrates.
Main Results:
- Replacement of leucine 301 with serine, glutamic acid, proline, or threonine induced ligand-independent transforming activity.
- Serine, glutamic acid, and proline mutations were more potent than threonine.
- Mutant CSF-1R variants S301 and E301 showed partially inhibited intracellular transport.
- Mutations at codon 301 led to constitutive CSF-1R kinase activity and increased tyrosine phosphorylation of cellular substrates.
Conclusions:
- Specific mutations at codon 301 of CSF-1R can mimic ligand binding, leading to constitutive activation of its growth-promoting activity.
- Altered intracellular transport and constitutive kinase activity contribute to CSF-1R-mediated cell transformation.
- These findings provide insights into the oncogenic potential of CSF-1R mutations.
