Complex CSF3R Protein Isoform Interactions Dictate Cytokine Responsiveness

CSF3R, the receptors for granulocyte colony stimulating factor, is a critical regulator of neutrophil production. Multiple CSF3R mRNA transcripts have been identified and are annotated in Genbank. The expression and function of the different CSF3R proteins have not been fully elucidated. We generated antibodies specific for two of the identified and annotated isoforms, V3 and V4. CSF3R-V4 is a truncated variant of V1 with a unique C-terminal 34 amino acids and this variant confers enhanced growth signals. Changes in the ratio of V1:V4 isoforms have been implicated in chemotherapy resistance and relapse of AML. CSF3R-V3 is a variant of V1 with a 27 amino acid insertion between two conserved domains in the cytoplasmic portion of the receptor involved in JAK/STAT activation, termed the box 1 and box 2. CSF3R-V3 produces reduced proliferative signaling in response to G-CSF. When V3 is co-expressed with V1, proliferative signaling is reduced in a concentration dependent manner. In order to generate custom rabbit polyclonal antibodies specific for CSF3R-V3 and CSF3R-V4 we used either a peptide that corresponds to a unique amino acid sequence present only in CSF3R-V3 or a peptide specific for a portion of the C-terminal amino acid sequence unique to the CSF3R-V4 isoform conjugated to an immunogenic carrier protein. These immunogens both produced robust immune responses, and the polyclonal antibodies were subsequently purified from bulk sera. Immunoblot analysis of lysates from Ba/F3 cells expressing CSF3R-V1 (V1), CSF3R-V3 (V3), or CSF3R-V4 (V4) demonstrated that both the custom generated anti-CSF3R-V3 and anti-CSF3R-V4 antibodies were very specific, recognizing only the appropriate CSF3R receptor isoform. All three CSF3R splice variants are recognized by commercially available anti-CSF3R (clone LMM741 to CD114), while the anti-CSF3R-V4 custom antibody and the custom anti-CSF3R-V3 antibody recognizes only the CSF3R-V4 and CSF3R-V3 isoforms, respectively. We next sought to detect the CSF3R receptor isoforms in primary human cells. Using our custom antibodies, we detected for the first time, both the CSF3R-V3 and CSF3R-V4 receptor forms in primary neutrophils isolated from healthy donors. Each of the CSF3R isoforms produce unique signaling, and we hypothesized that the observed differences in G-CSF-dependent signaling is produced by the expression level of each receptor isoform via both homodimerization and by heterodimerization of the receptor splice variant proteins. To investigate the potential for heterodimerization of the CSF3R-V1 with the V3 and V4 isoforms, we generated a CSF3R-V1 with a c-terminal epitope tag and co-expressed this construct with both CSF3R-V3 or CSF3R-V4. Immunoprecipitation with an antibody to the epitope tag (recognizing the V1 variant) followed by immunoblotting with the custom anti-V3 or anti-V4 antibodies demonstrated that both CSF3R-V3 and CSF3R-V4 co-immunoprecipitated with CSF3R-V1, in agreement with our hypothesis that the splice variants form receptor heterodimers. Of note, the CSF3R receptor heterodimers are detected even in the absence of G-CSF, thus demonstrating that CSF3R exist as a preformed receptor dimer in an inactive state. In conclusion, we have generated antibodies that specifically detect the CSF3R-V3 and the CSF3R-V4 receptor proteins. These are the first studies to demonstrate the expression of the CSF3R splice variants at the protein level, in both cell lines and primary human cells. In addition, these are the first studies to demonstrate the formation of heterodimers of the CSF3R splice variants, providing a mechanism for the observed alteration in ligand-dependent signaling produced under conditions of altered splice variant expression. Disclosures Avalos: Juno: Membership on an entity's Board of Directors or advisory committees; Best Practice-Br Med J: Patents & Royalties: receives royalties from a coauthored article on evaluation of neutropenia.

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Streptococcus salivarius Fimbriae Are Composed of a Glycoprotein Containing a Repeated Motif Assembled into a Filamentous Nondissociable Structure

Journal of Bacteriology ◽

10.1128/jb.183.9.2724-2732.2001 ◽

2001 ◽

Vol 183 (9) ◽

pp. 2724-2732 ◽

Cited By ~ 22

Author(s):

Céline Lévesque ◽

Christian Vadeboncoeur ◽

Fatiha Chandad ◽

Michel Frenette

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Cell Surface ◽

Polyclonal Antibodies ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Spontaneous Mutant ◽

Streptococcus Salivarius ◽

Streptococcal Species ◽

Repeated Motif

ABSTRACT Streptococcus salivarius, a gram-positive bacterium found in the human oral cavity, expresses flexible peritrichous fimbriae. In this paper, we report purification and partial characterization of S. salivarius fimbriae. Fimbriae were extracted by shearing the cell surface of hyperfimbriated mutant A37 (a spontaneous mutant of S. salivarius ATCC 25975) with glass beads. Preliminary experiments showed that S. salivariusfimbriae did not dissociate when they were incubated at 100°C in the presence of sodium dodecyl sulfate. This characteristic was used to separate them from other cell surface components by successive gel filtration chromatography procedures. Fimbriae with molecular masses ranging from 20 × 106 to 40 × 106Da were purified. Examination of purified fimbriae by electron microscopy revealed the presence of filamentous structures up to 1 μm long and 3 to 4 nm in diameter. Biochemical studies of purified fimbriae and an amino acid sequence analysis of a fimbrial internal peptide revealed that S. salivarius fimbriae were composed of a glycoprotein assembled into a filamentous structure resistant to dissociation. The internal amino acid sequence was composed of a repeated motif of two amino acids alternating with two modified residues: A/X/T-E-Q-M/φ, where X represents a modified amino acid residue and φ represents a blank cycle. Immunolocalization experiments also revealed that the fimbriae were associated with a wheat germ agglutinin-reactive carbohydrate. Immunolabeling experiments with antifimbria polyclonal antibodies showed that antigenically related fimbria-like structures were expressed in two other human oral streptococcal species, Streptococcus mitis andStreptococcus constellatus.

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Expression of Na+-d-glucose cotransporter in brush-border membrane of the chicken intestine

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1999.276.2.r627 ◽

1999 ◽

Vol 276 (2) ◽

pp. R627-R631 ◽

Cited By ~ 13

Author(s):

Carles Garriga ◽

Nativitat Rovira ◽

Miquel Moretó ◽

Joana M. Planas

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Sequence ◽

Western Blot ◽

Brush Border ◽

Synthetic Peptide ◽

Brush Border Membrane ◽

Polyclonal Antibodies ◽

Membrane Vesicles ◽

Dependent Transport

We have studied the expression of Na+-d-glucose cotransporter in brush-border membrane vesicles (BBMVs) of chicken enterocytes to correlate the changes in the apical Na+-dependent transport with the changes in the amounts of transporter determined by Western blot analysis. Two different rabbit polyclonal antibodies were used simultaneously. The antibody raised against amino acids 564–575 of the deduced amino acid sequence of rabbit intestinal SGLT-1 ( antibody 1) specifically detects a single 75-kDa band in the three segments, and this band disappeared when the antibody was preabsorbed with the antigenic peptide. The antibody raised against the synthetic peptide corresponding to amino acids 402–420 of the same protein ( antibody 2) only reacts with jejunal and ileal samples, but no signal is found in BBMVs of rectum. Only when antibody 1 was used was there a linear correlation between the maximal transport rates of hexoses in BBMVs and the relative protein amounts determined by Western blot. These results indicate that the Na+-d-glucose cotransport in the jejunum, the ileum, and the rectum of chickens is due to an SGLT-1 type protein.

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An Alternative Splice Variant of HIPK2 with Intron Retention Contributes to Cytokinesis

Cells ◽

10.3390/cells9020484 ◽

2020 ◽

Vol 9 (2) ◽

pp. 484 ◽

Cited By ~ 1

Author(s):

Veronica Gatti ◽

Manuela Ferrara ◽

Ilaria Virdia ◽

Silvia Matteoni ◽

Laura Monteonofrio ◽

...

Keyword(s):

Cell Division ◽

Stress Response ◽

Alternative Splice ◽

Functional Activity ◽

Splice Variant ◽

Splice Variants ◽

Stop Codon ◽

Cellular Stress Response ◽

Dependent Manner ◽

Western Blots

HIPK2 is a DYRK-like kinase involved in cellular stress response pathways, development, and cell division. Two alternative splice variants of HIPK2, HIPK2-FL and HIPK2-Δe8, have been previously identified as having different protein stability but similar functional activity in the stress response. Here, we describe one additional HIPK2 splice variant with a distinct subcellular distribution and functional activity in cytokinesis. This novel splice variant lacks the last two exons and retains intron13 with a stop codon after 89 bp of the intron, generating a short isoform, HIPK2-S, that is detectable by 2D Western blots. RT-PCR analyses of tissue arrays and tumor samples show that HIPK2-FL and HIPK2-S are expressed in normal human tissues in a tissue-dependent manner and differentially expressed in human colorectal and pancreatic cancers. Gain- and loss-of-function experiments showed that in contrast to HIPK2-FL, HIPK2-S has a diffuse, non-speckled distribution and is not involved in the DNA damage response. Rather, we found that HIPK2-S, but not HIPK2-FL, localizes at the intercellular bridge, where it phosphorylates histone H2B and spastin, both required for faithful cell division. Altogether, these data show that distinct human HIPK2 splice variants are involved in distinct HIPK2-regulated functions like stress response and cytokinesis.

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Characterization of Dendritic Cell-Specific Genes p275 and p306.

Blood ◽

10.1182/blood.v108.11.3842.3842 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3842-3842

Author(s):

Ingo Hilgendorf ◽

Daniel Kurz ◽

Anita Bringmann ◽

Lothar Kanz ◽

Frank Grünebach ◽

...

Keyword(s):

Peripheral Blood ◽

Splice Variant ◽

Splice Variants ◽

Polyclonal Antibodies ◽

Antigen Presenting Cells ◽

Poly I:C ◽

Blood Monocytes ◽

Splice Form ◽

Antigen Presenting

Abstract Dendritic cells play an inimitable role in the functioning immune system as they are the most potent antigen presenting cells being able to prime naive T-cells. Their characteristic properties that enable them to take up antigens and present them to leukocytes are due to an expression of specific genes and thus specific proteins that are unique to this subset of antigen-presenting cells. Using a substractive cDNA library based on suppression hybridization between DC cDNA and the reference monocyte cDNA, we identified in DC two differentially expressed genes p275 and p306. p275 codes for a membrane protein and represents a splice isoform of the transport protein NAT-1. The predicted structure of protein p306 is globular, suggesting that the protein is either intracellular or secreted. The expression of both genes was confirmed by RT-PCR using cDNA isolated from peripheral blood monocytes and DC, generated in vitro from monocytes or CD34+ progenitor cells. To further analyze the protein expression polyclonal antibodies were generated by immunization with synthetic peptides deduced from the identified sequences. Interestingly, inhibition of DC differentiation using IL-10 or STI571 (Imatinib) resulted in an impaired expression of both proteins. Utilizing specific primers for two recently described splice variants of p306 we identified a new splice form expressed in DC. While the gene of p306 contains eight exons, splice variant 1 consists of the exons 1,2,4,5,6, and 7 and splice variant 2 contains the exons 1,2,3,4,5,6, a shortened exon 7, and exon 8. The new identified splice form includes the exons 1–7. However, as the open reading frame starts in exon 4, the expressed protein is identical with the one corresponding to splice variant 1. Analyzing different DC populations in peripheral blood we show that p306 is expressed in plasmacytoid, but not myeloid DC. Interestingly, the activation of DC with Toll-like receptor ligands (TLRL) Pam3Cys (TLR2L), Poly I:C (TLR3L), LPS (TLR4L) and R848 (TLR7L) has no influence on the expression of p306. Although the functions of p275 and p306 in DC have yet to be determined, both genes play a role in DC differentiation and are found in different hematopoietic cell populations. Especially p306 might be an interesting marker of plasmacytoid DC as the predicted protein does not resemble any known protein structure.

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Two novel phosphatidylinositol-4-phosphate 5-kinase type Iγ splice variants expressed in human cells display distinctive cellular targeting

Biochemical Journal ◽

10.1042/bj20090638 ◽

2009 ◽

Vol 422 (3) ◽

pp. 473-482 ◽

Cited By ~ 51

Author(s):

Nicholas J. Schill ◽

Richard A. Anderson

Keyword(s):

Amino Acids ◽

Splice Variant ◽

Protein Targeting ◽

Splice Variants ◽

Human Cells ◽

Subcellular Targeting ◽

Splice Isoforms ◽

Messenger Molecules ◽

Phosphatidylinositol 4

The generation of various phosphoinositide messenger molecules at distinct locations within the cell is mediated via the specific targeting of different isoforms and splice variants of phosphoinositide kinases. The lipid messenger PtdIns(4,5)P2 is generated by several of these enzymes when targeted to distinct cellular compartments. Several splice variants of the type Iγ isoform of PIPK (PtdIns4P 5-kinase), which generate PtdIns(4,5)P2, have been identified, and each splice variant is thought to serve a unique functional role within cells. Here, we have identified two novel C-terminal splice variants of PIPKIγ in human cells consisting of 700 and 707 amino acids. These two splice variants are expressed in multiple tissue types and display PIPK activity in vitro. Interestingly, both of these novel splice variants display distinct subcellular targeting. With the addition of these two new splice isoforms, there are minimally five PIPKIγ splice variants that have been identified in mammals. Therefore, we propose the use of the HUGO (Human Genome Organization) nomenclature in the naming of the splice isoforms. PIPKIγ_i4 (700 amino acids) is present in the nucleus, a targeting pattern that has not been previously observed in any PIPKIγ splice variant. PIPKIγ_i5 (707 amino acids) is targeted to intracellular vesicle-like structures, where it co-localizes with markers of several types of endosomal compartments. As occurs with other PIPKIγ splice variants, the distinctive C-terminal sequences of PIPKIγ_i4 and PIPKIγ_i5 may facilitate association with unique protein targeting factors, thereby localizing the kinases to their appropriate cellular subdomains for the site-specific generation of PtdIns(4,5)P2.

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The mammalian ARF-like protein 1 (Arl1) is associated with the Golgi complex

Journal of Cell Science ◽

10.1242/jcs.109.1.209 ◽

1996 ◽

Vol 109 (1) ◽

pp. 209-220 ◽

Cited By ~ 12

Author(s):

S.L. Lowe ◽

S.H. Wong ◽

W. Hong

Keyword(s):

Amino Acid ◽

Golgi Complex ◽

Polyclonal Antibodies ◽

Brefeldin A ◽

Amino Acid Identity ◽

Northern Blotting ◽

Small Gtpase ◽

Rat Tissues ◽

Dependent Manner ◽

Additional Cell

A rat cDNA clone was isolated which encodes a protein displaying characteristics of a ras-like small GTPase. The deduced amino acid sequence shows the highest amino acid identity (79%) with the Drosophila ARF-like protein 1 (dArl1) among all the known members of the ras-like small GTPase superfamily. The encoded protein was tentatively named rat Arl1 (rArl1). Northern blotting analysis revealed that the rArl1 gene is ubiquitously expressed in rat tissues. Recombinant rArl1 fused to glutathione-S-transferase (GST) to create GST-rArl1 binds GTP-gamma-S in a dose-dependent manner. Polyclonal antibodies raised against two unique rArl1 peptides recognized a 22 kDa protein in total NRK cell lysate. Immunofluorescence microscopy of NRK cells revealed discrete perinuclear labelling that could be competed out by GST-rArl1 but not GST. Examination of 8 additional cell lines revealed a similar labelling, suggesting that the antigen recognised by the antibodies is conserved and widely-expressed. Co-localization experiments in NRK cells with antibodies to mannosidase II and a newly identified cis-Golgi protein, p28, showed that rArl1 is localized to the Golgi complex. When cells were treated with nocodazole, the Golgi complex marked by mannosidase II and p28 was fragmented into punctate structures scattered throughout the cell, in which rArl1 was colocalized. Treatment with brefeldin A (BFA) resulted in the redistribution of rArl1 and mannosidase II into the cytoplasm and endoplasmic reticulum, respectively. The kinetics of the redistribution of rArl1 in response to BFA differ from those of ARF and beta-COP, two components of non-clathrin coated vesicles.

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GS32, a Novel Golgi SNARE of 32 kDa, Interacts Preferentially with Syntaxin 6

Molecular Biology of the Cell ◽

10.1091/mbc.10.1.119 ◽

1999 ◽

Vol 10 (1) ◽

pp. 119-134 ◽

Cited By ~ 54

Author(s):

Siew Heng Wong ◽

Yue Xu ◽

Tao Zhang ◽

Gareth Griffiths ◽

Stephen Loucian Lowe ◽

...

Keyword(s):

Amino Acid ◽

Golgi Apparatus ◽

Amino Acid Sequence ◽

Northern Blot Analysis ◽

Amino Acid Sequence Identity ◽

Biochemical Characterization ◽

Polyclonal Antibodies ◽

Sequence Identity ◽

Membrane Bound

Syntaxin 1, synaptobrevins or vesicle-associated membrane proteins, and the synaptosome-associated protein of 25 kDa (SNAP-25) are key molecules involved in the docking and fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, cell biological, and biochemical characterization of a 32-kDa protein homologous to both SNAP-25 (20% amino acid sequence identity) and the recently identified SNAP-23 (19% amino acid sequence identity). Northern blot analysis shows that the mRNA for this protein is widely expressed. Polyclonal antibodies against this protein detect a 32-kDa protein present in both cytosol and membrane fractions. The membrane-bound form of this protein is revealed to be primarily localized to the Golgi apparatus by indirect immunofluorescence microscopy, a finding that is further established by electron microscopy immunogold labeling showing that this protein is present in tubular-vesicular structures of the Golgi apparatus. Biochemical characterizations establish that this protein behaves like a SNAP receptor and is thus named Golgi SNARE of 32 kDa (GS32). GS32 in the Golgi extract is preferentially retained by the immobilized GST–syntaxin 6 fusion protein. The coimmunoprecipitation of syntaxin 6 but not syntaxin 5 or GS28 from the Golgi extract by antibodies against GS32 further sustains the preferential interaction of GS32 with Golgi syntaxin 6.

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An unusual fibrillarin gene and protein: structure and functional implications.

Molecular Biology of the Cell ◽

10.1091/mbc.8.6.1051 ◽

1997 ◽

Vol 8 (6) ◽

pp. 1051-1061 ◽

Cited By ~ 14

Author(s):

E David ◽

J B McNeil ◽

V Basile ◽

R E Pearlman

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Polyclonal Antibodies ◽

Single Copy ◽

Rrna Processing ◽

Dna Encoding ◽

Yeast Saccharomyces Cerevisiae ◽

Cis Acting ◽

Functional Conservation ◽

Functional Implications

The diploid germinal nucleus of the ciliated protozoan Tetrahymena thermophila is unusual among eukaryotes in that it encodes a single copy of the gene for rRNA allowing identification of cis-acting mutations in rDNA affecting rRNA structure, function, and processing. The generally conserved nucleolar protein fibrillarin has been characterized from a number of systems and is involved in pre-rRNA processing. We have demonstrated that Tetrahymena has fibrillarin and have analyzed the cDNA and the genomic DNA encoding this protein. The derived amino acid sequence of the N-terminal region of Tetrahymena fibrillarin shows little similarity with the generally highly conserved glycine/arginine-rich N-terminal domain of other eukaryotic fibrillarins. The remainder of the amino acid sequence of the molecule is more conserved. Polyclonal antibodies generated against the full-length Tetrahymena fibrillarin expressed in bacteria recognize a protein of M(r) approximately 32,000 in whole-cell or nucleolar preparations. Immunocytochemistry localizes fibrillarin to nucleoli in the somatic macronuclei of vegetative cells. Transformation experiments demonstrate that fibrillarin is an essential protein in Tetrahymena. The Tetrahymena fibrillarin is expressed but does not complement a NOP1 null mutation when transformed into the yeast Saccharomyces cerevisiae, indicating less functional conservation among fibrillarins than previously suggested.

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Brevinin-1 and multiple insulin-releasing peptides in the skin of the frog Rana palustris

Journal of Endocrinology ◽

10.1677/joe.0.1810347 ◽

2004 ◽

Vol 181 (2) ◽

pp. 347-354 ◽

Cited By ~ 28

Author(s):

L Marenah ◽

PR Flatt ◽

DF Orr ◽

S McClean ◽

C Shaw ◽

...

Keyword(s):

Mass Spectrometry ◽

Amino Acid ◽

Amino Acid Sequence ◽

Insulin Release ◽

Biologically Active ◽

Mass Spectrometry Analysis ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Bicarbonate Buffer ◽

Skin Secretions

Few studies have comprehensively examined amphibian granular gland secretions for novel insulinotropic peptides. This study involved isolation and characterisation of biologically active peptides from the skin secretions of Rana palustris frogs. Crude secretions obtained by mild electrical stimulation from the dorsal skin surface were purified by reversed-phase HPLC on a semipreparative Vydac C18 column, yielding 80 fractions. These fractions were assayed for insulin-releasing activity using glucose-responsive BRIN-BD11 cells. Acute 20 min incubations were performed in Krebs Ringer bicarbonate buffer supplemented with 5.6 mmol/l glucose in the absence (control) and presence of various fractions. Fractions 29-54 and fractions 68-75 showed significant 2.0-6.5-fold increases in insulin-releasing activity (P<0.001). The fractions showing most prominent insulinotropic activity were further purified to single homogeneous peaks, which, on testing, evoked 1.5-2.8-fold increases in insulin release (P<0.001). The structures of the purified peptides were determined by mass spectrometry and N-terminal amino acid sequencing. Electrospray ionisation ion-trap mass spectrometry analysis revealed molecular masses of 2873.5-8560.4 Da. Sufficient material was isolated to determine the primary amino acid sequence of the 2873.5 Da peptide, revealing a 27 amino acid sequence, ALSILRGLEKLAKMGIALTNCKATKKC, repressing palustrin-1c. The database search for this peptide showed a 48% homology with brevinin-1, an antimicrobial peptide isolated from various Rana species, which itself stimulated insulin release from BRIN-BD11 cells in a concentration-dependent manner. In conclusion, the skin secretions of R. palustris frogs contain a novel class of peptides with insulin-releasing activity that merit further investigation.

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CSF3R Splicing Regulates Granulopoiesis Via Splice Variant Specific Responses to G-CSF

Blood ◽

10.1182/blood-2021-154322 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3130-3130

Author(s):

Amanda Lance ◽

Rajeswaran Mani ◽

Sara L. Seegers ◽

Belinda R Avalos ◽

Lawrence J Druhan

Keyword(s):

Cell Proliferation ◽

Splice Variant ◽

Splice Variants ◽

Myeloid Cell ◽

Model System ◽

Lymphoid Cells ◽

Dependent Manner ◽

Wild Type ◽

Knock Out ◽

Dose Dependent

Abstract The granulocyte colony stimulating factor receptor (CSF3R) is a critical regulator of neutrophil production with multiple alternatively spliced variants. The truncated CSF3R-V4 splice variant confers enhanced growth signals, and changes in its expression levels relative to the canonical V1 (wild type) isoform have been implicated in chemotherapy resistance and relapse of AML. We previously demonstrated that the CSF3R-V3 isoform, a variant of V1 with an insertion in the cytoplasmic domain, produces hypoproliferative signals in lymphoid cells in response to G-CSF. We also reported that expression of all three splice variants is significantly altered in AML, suggesting that aberrant CSF3R splicing is involved in the pathogenesis of some myeloid malignancies. The functional signaling capabilities of the different CSF3R isoforms in regulating granulopoiesis remain largely unknown. Herein, we describe a novel myeloid model system and show that the V3 and V4 isoforms generate opposing proliferative signals without effects on myeloid cell differentiation. ER-HoxB8 cells are murine bone marrow progenitor cells ectopically expressing an ER-HoxB8 fusion protein, and in the presence of estradiol (E2) the fusion protein dimerizes producing a functional HoxB8 dimer which enforces self-renewal. Thus, in the presence of E2 these cells continually proliferate; however, when E2 is withdrawn they differentiate into mature granulocytes. Addition of G-CSF to culture medium of E2 ER-HoxB8 cells increases progenitor cell proliferation in a dose dependent manner (Figure 1A). Using CRISPR/Cas9, we knocked-out the endogenous murine Csf3r. As expected, ER-HoxB8-Csf3r-/- cells still produced mature neutrophils with E2 withdrawal and no increase in differentiation or proliferation of the knock-out cells (KO) was observed in response to G-CSF. The functional behavior of our ER-HoxB8-Csf3r-/- cells recapitulates the published phenotype of the Csf3r knock-out mouse, which exhibits severe neutropenia but has circulating neutrophils. ER-HoxB8 KO cells were transduced with human CSF3R splice variants and expression confirmed by immunoblot analysis using splice-variant specific antibodies. KO cells expressing the CSF3R-V3 demonstrated a hypoproliferative response to G-CSF with an ~40-fold increase in the EC50 relative to cells expressing CSF3R-V1 (Figure 1B), confirming our prior observations in the lymphoid BaF3 cell line. In contrast, KO cells expressing the truncated CSF3R-V4 variant hyperproliferated in response to G-CSF consistent with our previously published data in lymphoid cells. Using multi-color flow cytometry with antibodies against CD117, CD11b, and Ly6G to identify progenitor, intermediately differentiated cells (NeuP), and mature neutrophils, we found that KO cells (like parental ER-HoxB8 cells) produced significant numbers of CD11b+/LyGG- NeuP cells upon E2 withdrawal and addition of G-CSF had no effect on differentiation. Transduction of ER-HoxB8 KO cells with the wild type human CSF3R-V1 restored their capacity to respond to G-CSF in a dose dependent manner. KO cells transduced with CSF3R-V3 displayed normal production of NeuP cells with E2 withdrawal, and addition of G-CSF produced a substantial increase in the numbers of mature neutrophils (CD11b+, Ly6G+) after 5 days in culture, relative to KO cells (Figure 2C). Thus, we have demonstrated that CSF3R-V3 is able to support the production of fully mature neutrophils. Notably, a G-CSF induced increase in the numbers of mature neutrophils was also evident in CSF3R-V4 transduced cells. Previous work by others indicated that CSF3R-V4 was not able to drive myeloid differentiation. We hypothesize that this difference in phenotype is due to a V4-dependent hyperproliferation of the neutrophil progenitors. On-going work is focused on the determination of the specific effects these CSF3R splice variants have on each stage of granulopoiesis. In conclusion, using our novel engineered CSF3R model system, we confirm differential effects of CSF3R splice variants on myeloid cell proliferation and show sustained differentiation capacity of each isoform. Additional studies using this model system provide the opportunity for identification of new therapeutic targets for treatment of disorders of granulocyte production. Figure 1 Figure 1. Disclosures Avalos: JUNO: Membership on an entity's Board of Directors or advisory committees.

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