Characterization of G-CSF Receptor Signaling Pathways

Leukemia is a hematological cancer that specifically affects neutrophils—the primary white blood cells and the first line of defense in the body. G-CSF, granulocyte colony-stimulating factor, supports the proliferation of early myeloid progenitor cells and the maturation of these cells to granulocytes. Myeloid progenitor cells are equipped with receptors on their cell surfaces in order to respond to various cytokines. Loss-of-function mutations in the G-CSF receptor lead to impaired granulopoiesis. In contrast, a mutation leading to a truncated G-CSFR (termed d715, see below) results in a hyperproliferative response to G-CSF and is associated with acute leukemia.

Little is known about the G-CSF pathway, other than that it stimulates myeloid cells to differentiate into white blood cells. The Link Lab is interested in understanding how the pathway works with the primary question being what proteins and pathways are activated when stimulated with G-CSF at various time points? Once this is understood, the up- or down- regulation of neutrophils can be better understood and possibly controlled.

There are five receptor types used in my project to study the G-CSF pathway: wild type, knockout, d715, d715F, and G:Epo. Wild type is a normal receptor. Knockout is the absence of a receptor. d715 is a truncated G-CSF receptor and is found in approximately 20% of patients with severe congenital neutropenia, a syndrome manifested by a severe decrease in the number of neutrophils. d715F is an engineered receptor where the sole remaining tyrosine of d715 has been mutated to phenylalanine, causeing neutropenia. G:Epo is a hybrid receptor where the entire cytoplasmic domain of the G-CSFR is replaced with that of the erythropoietin receptor (EpoR).

The goal of my project was to characterize signaling pathways in mutant receptors. To do so, the d715 and d715F mutant receptor DNA had to be cut out of the old pBS vector and ligated into the new pBOS vector. A plasmid mini-prep was then performed to screen clones for the plasmid. Once this was accomplished, the mutant plasmids were cotransfected along with the PGK-NEO selection cassette by electroporating the BAF cells. Colonies were selected based on their ability to grow in G418 containing media. Expression of functional G-CSFR on these clones was analyzed by flow cytometry to detect the binding of biotinylated-G-CSF. To date, we have identified BAF clones expressing wild type G-CSFR and the 3 G-CSFR mutants. Studies are underway to characterize the signaling pathways activated by these receptors.