“Colony-stimulating factors” were first described in the 1960s as soluble agents that mediated the growth of colonies in soft agar from bone marrow cells. One of these was called granulocyte colony-stimulating factor (G-CSF) due to its propensity to induce colonies composed of neutrophilic granulocytes. The purification, sequencing, and cloning of this factor led to the production of recombinant G-CSF that facilitated the identification of a specific cell-surface receptor through expression library screening (Fukunaga et al. 1990). This protein was named granulocyte colony-stimulating factor receptor (G-CSFR), although the gene has now been officially designated colony-stimulating factor 3 receptor (CSF3R).
G-CSFR Evolution and Structure
G-CSFR is a member of the class I cytokine receptor superfamily. These molecules emerged early in bilaterian evolution with a protein structurally very similar to G-CSFR. Subsequent duplication of this precursor yielded the G-CSFR, which is found throughout higher vertebrates, from fish to humans (Liongue et al. 2016).
Signal Transduction Mediated by G-CSFR and Its Control
G-CSF binds to G-CSFR in a 2:2 or 4:4 stoichiometry, which causes structural alterations in the receptor complex that are transmitted through to the cytoplasmic domain. This leads to activation of multiple tyrosine kinases associated with the intracellular domain of the receptor, including members of the Janus kinases (JAK) family, especially JAK1 and JAK2, SRC kinases such as LYN and HCK, as well as SYK and TNK. These kinases then activate multiple downstream signaling molecules: signal transducer and activator of transcription (STAT) family members, especially STAT3 and STAT5, as well as components of the phosphatidyl inositol 3-kinase (PI3-K)-AKT and RAS-MAPK pathways. A number of these molecules are recruited by binding to specific tyrosine residues on the receptor (or associated components) that become phosphorylated in the process (Touw and van de Geijn 2007) (Fig. 1).
Signaling via the G-CSFR is tightly regulated, with maximal signaling reached quickly post stimulation after which it is rapidly extinguished. This is achieved by a combination of negative regulatory processes. These include direct regulation by the tyrosine phosphatases SHP-1 and SHP-2, which are also recruited to the activated receptor complex. Additional regulation in the form of a negative feedback loop is afforded by members of the suppressor of cytokine signaling (SOCS) family, especially SOCS3 and CISH, which are induced by G-CSFR signaling and then dock to the receptor (Touw and van de Geijn 2007). SOCS3 is particularly important and acts to facilitate ubiquitination of a lysine residue adjacent to Box 1 that works in concert with a dileucine internalization motif within Box 3 to facilitate receptor degradation (Palande et al. 2013).
Biological Functions of G-CSFR
One major role that is conserved throughout higher vertebrates is during myelopoiesis, where G-CSFR is especially important in the production of neutrophilic granulocytes during “emergency” situations. However, it further acts at the granulocyte-macrophage progenitor (GMP) level and so also affects the monocyte/macrophage lineage (Liongue et al. 2009a; Liu et al. 1996). G-CSFR signaling additionally mediates the effective mobilization of mature neutrophilic granulocytes and HPCs from the bone marrow via trans-acting signals that are independent of G-CSFR expression (Greenbaum and Link 2011). G-CSFR further influences the motility – including directional migration – of mature neutrophils as well as myeloid progenitors and can also contribute to the activation of neutrophils (Liongue et al. 2009b).
G-CSFR signaling has been implicated outside the hematopoietic system as well, particularly in tissue protection and repair (Liongue et al. 2009b). For example, G-CSF is neuroprotective, directly stimulating neurogenesis and inhibiting apoptosis, but also suppressing inflammation and mobilizing bone marrow stem cells to the brain, thereby improving neural plasticity (Klocke et al. 2008). It also can augment the repair and regeneration of both skeletal and cardiac muscle (Klocke et al. 2008; Pitzer et al. 2008).
Role of G-CSFR in Disease
“Crippling” Extracellular Mutations: Either large deletions (e.g., p. Ser319Gly, fs*29) or mutations of critical structural residues (e.g., p. Pro229His) in the extracellular domain that severely disrupt downstream signalling, with loss of STAT5 activation particularly important (Ward et al. 1999). These mutations, which can be inherited or acquired, have been associated with severe congenital neutropenia (SCN) and chronic idiopathic neutropenia (CIN) (Liongue and Ward 2014; Touw and Beekman 2013).
“Activating” Transmembrane Mutations: Typically single amino substitutions within the transmembrane domain or adjacent residues (e.g., p. Thr618Ile). This class of mutations act in a dominant manner by stabilizing transmembrane helix-helix interactions resulting in G-CSF-independent activation, growth, and differentiation (Maxson et al. 2013). Such mutations are commonly acquired in chronic neutrophilic leukemia (CNL) but additionally in atypical chronic myelogenous leukemia (aCML), chronic myelomonocytic leukemia (CMML), early T-cell precursor acute lymphoblastic leukemia (ETP-ALL), and de novo acute myeloid leukemia (AML). A heritable mutant (p. Thr617Asn) has been identified as the cause of autosomal dominant hereditary neutrophilia, with one affected individual carrying this mutation progressing to myelodysplastic syndrome (MDS) (Liongue and Ward 2014; Touw and Beekman 2013).
“Hyperresponsive” Intracellular Truncations: Acquired truncations of the carboxyl-terminus (e.g., p. Gln741*, p. Gln754*) which act dominantly over wild-type receptors to mediate heightened growth and reduced maturation in response to ligand (Dong et al. 1995a). This results from sustained activation caused by a heavily reduced “off-rate” due to loss of the dileucine internalization motif in Box 3 and recruitment sites for key negative regulators, including SHP-1, SHP-2, CISH, and SOCS3. The hyperproliferative function of truncated G-CSFRs seems largely due to greatly elevated STAT5 activation. Mouse models of the G-CSFR truncation exhibited a variable basal neutropenia, but a strong hyperresponsiveness to exogenous G-CSF, producing elevated neutrophil numbers as a result of increased myeloid progenitor proliferation. G-CSFR truncations were able to cooperate with other leukemic oncogenes in a G-CSF-dependent manner. Truncated G-CSFRs are commonly acquired in SCN and while not the primary cause of this disease, those patients carrying truncating G-CSFR mutations are strongly predisposed to both MDS and AML (Liongue and Ward 2014; Touw and Beekman 2013).
“Defective” Splice Variants: Altered splicing of the CSF3R transcript leading to the replacement of the normal C-terminus of the intracellular domain with 34 residues from an alternate reading frame. Such proteins are unable to transduce either proliferation or maturation signals suggesting interference with normal G-CSFR signaling (Dong et al. 1995b). Found in AML, these variants can result from cellular changes that lead higher levels of a minor CSF3R transcript (p. Val750Ala, fs*34), or from presumably somatic single base changes in CSF3R, to generate a cryptic splice-donor site in de novo AML (p. Val707Ala, fs*34) (Liongue and Ward 2014).
“Pathogenic” SNP: A single amino acid variant in the intracellular domain (p. Glu808Lys) found in ~6% of the population, which acts in a dominant-negative manner to reduce colony formation compared to the wild-type receptor (Wolfler et al. 2005). Individuals with this SNP are predisposed to high-risk MDS, with blasts from one individual who progressed to AML found to be homozygous for this allele (Liongue and Ward 2014).
“Aberrant” Activation: G-CSFR – often together with G-CSF – has been shown to be misexpressed in a diverse range of nonhematological malignancies, including bladder, squamous cell, and ovarian carcinomas and Ewing’s sarcoma. In several instances, the resultant aberrant G-CSFR signaling has been demonstrated to contribute to key cancer cell phenotypes, including proliferation, survival, migration/invasion, and chemoresistance, as well as exerting proangiogenic and immunosuppressive functions that might also contribute to tumorigenesis (Kumar et al. 2014; Liongue et al. 2009b).
Activation of endogenous G-CSFR through injection of recombinant G-CSF has been widely used in the treatment of neutropenic conditions – including congenital forms and those associated with chemotherapy – leading to a significant reduction in infection-related events (Sung and Dror 2007). G-CSF has also been extensively used for harvesting of HSCs from the peripheral blood, thereby overcoming the requirement for bone marrow extraction. There are also potential applications in regenerative medicine, including the treatment of myocardial infarct, skeletal muscle, and neuromuscular disorders (Klocke et al. 2008).
However, since G-CSFR mutations and/or dysregulated signaling contribute to malignancy, appropriate caution should be given to the use of G-CSF in settings where this may be relevant. For example, it has been proposed that the risk of leukemia in SCN patients increased with the degree of G-CSF therapy (Donadieu et al. 2005). On the other hand, a number of pharmacologic agents are now available that target signaling pathways downstream of the G-CSFR, which may be suitable for patients harboring G-CSFR mutations or in which G-CSFR signaling is dysregulated.
G-CSFR is important in the generation of neutrophilic granulocytes, particularly during emergency hematopoiesis. However, it also contributes more broadly to the development of other myeloid lineages as well as the mobilization and migration of hematopoietic progenitor and other myeloid cell populations. This has resulted in several important clinical applications for its ligand, G-CSF, especially to assist with hematopoietic recovery after chemotherapy and other ablative treatments. Functions for G-CSFR outside the hematopoietic system have also been characterized, which may underpin additional therapeutic applications for G-CSF. A number of G-CSFR mutations have been identified that are associated with a spectrum of myeloid disorders, including malignancy. Additionally, autocrine/paracrine stimulation of G-CSFR may contribute to the biology of solid tumors. Therefore, judicious use of G-CSF – and consideration of alternative therapy – is advised in these clinical situations.
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