G-CSF Receptor

G-CSF Receptor Shigekazu Nagata* Department of Genetics, Osaka University Medical School, 2-2 Yamada-oka Suita, Osaka, 565-0871, Japan * corresponding author tel: ‡81-6-6879-3310, fax: 81-6-6879-3319, e-mail: [email protected] DOI: 10.1006/rwcy.2000.20006.

SUMMARY

Alternative names

The G-CSF receptor is a type I membrane protein which belongs to the cytokine receptor superfamily, and is specifically expressed in mature neutrophils and neutrophilic precursors. Binding of G-CSF to G-CSFR induces its dimerization. The dimerized receptor transduces growth and differentiation signals which are mediated by the N-terminal and C-terminal halves of the G-CSFR cytoplasmic region. G-CSF activates JAK family kinases, which cause tyrosine phosphorylation of STAT family transcription factors. STATs then stimulate proliferation, and induce differentiation of neutrophilic precursor cells. Genetic defects in the G-CSFR gene cause severe neutropenia (Kostmann's syndrome).

G-CSFR is also known as CD114.

BACKGROUND

Structure G-CSFR is a type I membrane protein of about 130 kDa.

Main activities and pathophysiological roles G-CSFR transduces proliferation and differentiation signals into neutrophilic precursor cells, thus mediating the action of G-CSF (production of neutrophils, granulopoiesis) (Fukunaga et al., 1991).

Discovery The receptor for G-CSF was discovered as a membrane protein expressed in myeloid leukemia cells or neutrophilic granulocytes to which [125I]labeled G-CSF binds (Nicola and Metcalf, 1984). Murine G-CSFR was purified from the membrane fraction of mouse myeloid leukemia NFS60 cells that respond to G-CSF for proliferation (Fukunaga et al., 1990b), and its cDNA was isolated from an NFS60 cDNA library by expression cloning (Fukunaga et al., 1990a). The human counterpart was subsequently identified from a human placenta cDNA library by crosshybridization with mouse cDNA (Fukunaga et al., 1990c), or from a human neutrophil cDNA library by expression cloning (Larsen et al., 1990).

GENE

Accession numbers Human G-CSFR: M59818 (Fukunaga et al., 1990c; Larsen et al., 1990) Murine G-CSFR: M32699 (Fukunaga et al., 1990a)

Sequence See Figure 1.

1946 Shigekazu Nagata Figure 1 Human G-CSFR 1 GAAGCTGGAC 61 CAAGCAGGGG 121 GAAGTAACTT 181 GGGAAACTGC 241 GGAGTGCGGG 301 CTCCTGCATC 361 ACTGGGAGCA 421 ATCTATCATC 481 CTGGGGCAAC 541 CATACCCCAC 601 GGAGCCAGGA 661 GGGCAACTGT 721 CCACTGCTGC 781 GGCAGAGAAT 841 TGTGAAACTG 901 CCAGGCAGGC 961 GAAGTGTGAG 1021 CCTCCCCTTG 1081 CCTGCAGATA 1141 CCTGGAGCTG 1201 GAGGCAGCTG 1261 CAGCGGACGG 1321 CCTGCCCCTC 1381 GGAGGTGGCC 1441 CTCAGAAAGC 1501 CCTCTGGGTA 1561 CCTGGGCCCC 1621 AGCCACGGGG 1681 GACTCCCTTG 1741 AATGGCTCCC 1801 GCTGGAGTGG 1861 CTTCTGGACC 1921 CTTTGTCCTC 1981 CCAGGCTGGG 2041 GGAGCTACAC 2101 AACTGCCTGG 2161 CCCAGCTCAC 2221 GCTGCCCGGC 2281 GAAGCCGGTG 2341 CCAGACCTAT 2401 TGGCACCAGC 2461 AGGGCACTAT 2521 CAAGTCCTAT 2581 CCCAAGCCAG 2641 GATCCGGGTC 2701 TTGGGCCTGC 2761 TGACTAAAAA 2821 CTCCCAATCT 2881 GCTCCATCCA 2941 AAC

Nucleotide sequence for the human G-CSFR gene.

TGCAGCTGGT CTGGGCCAGA GTCCAAGATC AGCCTGACTT CACATCAGTG ATCAAGCAGA GAGCTTCAGC ACCCTGCCCC AGCCTGCAGA AACCTCTCCT CCTGAGACCC CAGACCCAAG ATCCCACGCA GCGCTGGGGA GAGCCCCCCA TGCCTACAGC CTGCGCCACA GAGGCCCTTC CGCTGCATCC AGAACTACCG GACCCCAGGA ATCCAAGGTT TGCAACACCA CTTGTGGCCT AGAGGCCCAG GGCTGGGAGC CCCAGCGCGA TTTCTGCTGA TACCAGGACA TCCCATGCCC GTGCCTGAGC AACGCTCAGA CATGGCCTGG GCCACCAACA ATCATCCTGG CTCTGTTGCA AGCAGCCTGG CTTGGCACGC CCCTGGGAGT GTGCTCCAGG GATCAGGTCC CTCCGCTGTG GAGAACCTCT GAGGACGACT CATGGGATGG CTCTTAAAGG CTACCCCAGC CCATAGGCTG GCCCCACCCA

TTCAGGAACT GGTGCCAACA ACAAAGCTGG GGGCTGCCCT TCTCAGCCCC ACTGCAGCCA CCGGGGGCAG ACCTCAACCA TCCTGGACCA GCCTCATGAA ACCTACCCAC GGGACTCCAT AACACCTGCT CCAGCATGTC TGCTGCGGAC TGTGCTGGGA AGCCGCAGCG AGTATGAGCT GCTGGCCCCT AACGGGCCCC CAGTGCAGCT ATGTGGTTTC CAGAGCTCAG ATAACTCAGC CTCTGACCAG CCCCCAATCC GCAATAGCAA AGGAGAACAT CCATGGGACC CAGAGCTGCA CCCCTGAGCT ACCAGTCCTT AGCCCGCCAG GTACAGTCCT GCCTGTTCGG GCCCCAACAG GCTCCTGGGT CACCCATCAC CCCATAACAG GGGACCCAAG TTTATGGGCA ACTCCACTCA GGTTCCAGGC GTGTCTTTGG AGGCGCTGGG CCTGAGCTAG CCAGGCTCTC GGCCTCCCAG ATGGCCTTTT

TCTCTTGACG TGGGGAAACT TGAACATCAA GATCATCCTG CATCGTCCAC TCTGGACCCG GCAGCAGCGT CACTCAGGCC GGTTGAGCTG CCTCACAACC CAGCTTCACT CCTGGACTGC GTTGTACCAG CCCACAACTG CATGGACCCC GCCATGGCAG TGGAGAAGCC CTGCGGGCTC GCCTGGCCAC CACTGTCAGA GTTCTGGAAG TTGGAGACCC CTGCACCTTC CGGGACCTCT ACTCCATGCC ATGGCCTCAG CAAGACCTGG CAGGCCCTTT CTCCCAGCAT TCTAAAGCAC GGGGAAGAGC CTCCGCCATC TCTGTATCAC CACCCTGATG CCTCCTGCTG GAAGAATCCC GCCCACAATC CAAGCTCACA CTCAGAGACC AGCAGTTTCC GCTGCTGGGC GCCCCTCTTG CAGCCCCTTG GCCACTGCTC GAGCTTCTAG CTGGAGAAGA ACCATCTCCA GCGATCTGCA GTGCTTGTTT

AGAAGAGAGA GAGGCTCGGC GTTGGTGCTA CTGCTCCCCG CTGGGGGATC GAGCCACAGA CTGTCTGATG TTTCTCTCCT CGCGCAGGCT AGCAGCCTCA CTGAAGAGTT GTGCCCAAGG AATATGGGCA TGTCTTGATC AGCCCTGAAG CCAGGCCTGC AGCTGGGCAC CTCCCAGCCA TGGAGCGACT CTGGACACAT CCAGTGCCCC TCAGGCCAGG CACCTGCCTT CGCCCCACCC ATGGCCCGAG GGCTATGTGA AGGATGGAAC CAGCTCTATG GTCTATGCCT ATTGGCAAGA CCCCTTACCC CTGAATGCCT ATCCACCTCA ACCTTGACCC TTGCTCACCT CTCTGGCCAA ATGGAGGAGG GTGCTGGAGG TGTGGCCTCC ACCCAGCCCC AGCCCCACAA GCGGGCCTCA GGGACCCTGG AACTTCCCCC GGCTTCCTGG GGGGAGGGTC GTCACCAGCA TACTTTAAGG CCTATAACTT

CCAAGGAGGC TCGGAAAGGT TGGCAAGGCT GAAGTCTGGA CCATCACAGC TTCTGTGGAG GGACCCAGGA GCTGCCTGAA ACCCTCCAGC TCTGCCAGTG TCAAGAGCCG ACGGGCAGAG TCTGGGTGCA CCATGGATGT CGGCCCCTCC ACATAAATCA TGGTGGGCCC CGGCCTACAC GGAGCCCCAG GGTGGCGGCA TGGAGGAAGA CTGGGGCCAT CAGAAGCCCA CGGTGGTCTT ACCCTCACAG TTGAGTGGGG AGAATGGGAG AGATCATCGT ACTCTCAAGA CCTGGGCACA ACTACACCAT CCTCCCGTGG TGGCTGCCAG CAGAGGGGTC GCCTCTGTGG GTGTCCCAGA ATGCCTTCCA AGGATGAAAA CCACTCTGGT AATCCCAGTC GCCCAGGGCC CCCCCAGCCC TAACCCCAGC TCCTGCAGGG GGTTCCCTTC CATAAGCCCA TCTCCCTCTC ACCAGATCAT CAGTATTGTA

Chromosome location and linkages

Mouse G-CSFR: P40223 (Fukunaga et al., 1990a)

G-CSFR is on chromosome 1p35-34.3 in humans (Inazawa et al., 1991) and on chromosome 4 in the mouse (Ito et al., 1994).

Sequence

PROTEIN

Accession numbers Human G-CSFR: Q99062 (Fukunaga et al., 1990c; Larsen et al., 1990)

See Figure 2.

Description of protein Human G-CSFR comprises 836 amino acids with a signal sequence of 23 amino acids at the N-terminus (Fukunaga et al., 1990b). A single transmembrane

G-CSF Receptor 1947 Figure 2 Amino acid sequence for the human G-CSF receptor. Leader sequence is underlined and transmembrane domain is in bold and underlined. MARLGNCSLT ILWRLGAELQ YPPAIPHNLS DGQSHCCIPR AAPPQAGCLQ TAYTLQIRCI LEEDSGRIQG PVVFSESRGP QNGRATGFLL TWAQLEWVPE MAASQAGATN SVPDPAHSSL PTLVQTYVLQ TPSPKSYENL

WAALIILLLP PGGRQQRLSD CLMNLTTSSL KHLLLYQNMG LCWEPWQPGL RWPLPGHWSD YVVSWRPSGQ ALTRLHAMAR KENIRPFQLY PPELGKSPLT STVLTLMTLT GSWVPTIMEE GDPRAVSTQP WFQASPLGTL

GSLEECGHIS GTQESIITLP ICQWEPGPET IWVQAENALG HINQKCELRH WSPSLELRTT AGAILPLCNT DPHSLWVGWE EIIVTPLYQD HYTIFWTNAQ PEGSELHIIL DAFQLPGLGT QSQSGTSDQV VTPAPSQEDD

domain of 26 amino acids divides the molecule into the extracellular region of 604 amino acids, and the cytoplasmic region of 183 amino acids.

Relevant homologies and species differences Human and mouse G-CSF receptors have a homology of 62.5%, and there is no species specificity between human and mouse G-CSFRs (Nicola et al., 1985; Fukunaga et al., 1990c). The overall structure of GCSFR is similar to that of gp130 (the IL-6 receptor signal transducer) (Hibi et al., 1990) and LIF receptor (Gearing et al., 1991), and leptin receptor (Tartaglia et al., 1995). The ligand-binding domain (CRH domain, amino acids from 97 to 308 in mouse G-CSFR) in the extracellular region of G-CSFR (Fukunaga et al., 1991; Layton et al., 1997b) shows similarity to the corresponding region of other cytokine receptors (Bazan, 1990a,b; Fukunaga et al., 1990a,c). Two regions (box 1 and box 2) of the membrane-proximal region of G-CSFR are related to the corresponding regions of other cytokine receptors, while another region (box 3) is related to the corresponding region of gp130 (Fukunaga et al., 1991; Murakami et al., 1991).

Affinity for ligand(s) G-CSF binds to G-CSFR with Kd of about 100 pM (Fukunaga et al., 1990b; Nicola and Metcalf, 1984).

Cell types and tissues expressing the receptor G-CSFR is expressed in neutrophils, neutrophilic precursors in the bone marrow, myeloid leukemia cells, and placenta (Fukunaga et al., 1990a,c; Nicola and Metcalf, 1984).

VSAPIVHLGD HLNHTQAFLS HLPTSFTLKS TSMSPQLCLD KPQRGEASWA ERAPTVRLDT TELSCTFHLP PPNPWPQGYV TMGPSQHVYA NQSFSAILNA GLFGLLLLLT PPITKLTVLE LYGQLLGSPT CVFGPLLNFP

PITASCIIKQ CCLNWGNSLQ FKSRGNCQTQ PMDVVKLEPP LVGPLPLEAL WWRQRQLDPR SEAQEVALVA IEWGLGPPSA YSQEMAPSHA SSRGFVLHGL CLCGTAWLCC EDEKKPVPWE SPGPGHYLRC LLQGIRVHGM

NCSHLDPEPQ ILDQVELRAG GDSILDCVPK MLRTMDPSPE QYELCGLLPA TVQLFWKPVP YNSAGTSRPT SNSNKTWRME PELHLKHIGK EPASLYHIHL SPNRKNPLWP SHNSSETCGL DSTQPLLAGL EALGSF

Regulation of receptor expression Two regions in the promoter seem to be important for G-CSF receptor gene expression. C/EBP binds to a region located at ÿ49 from the transcription initiation site (Smith et al., 1996), which is essential for the expression of G-CSFR (Zhang et al., 1997). The other two cis-regulatory elements are located at ‡36 and ‡43, to which the ets family member PU.1. binds. Mutation of these sites reduces the promoter activity by 75% (Smith et al., 1996). However, mice lacking PU.1 can express G-CSFR (Olson et al., 1995), suggesting that this element may not be essential for the expression of the G-CSFR gene.

Release of soluble receptors An alternatively spliced mRNA coding for a protein lacking the transmembrane region can be detected in human U937 cells (Fukunaga et al., 1990c). However, the existence of soluble G-CSFR in human serum has not yet been reported.

SIGNAL TRANSDUCTION

Associated or intrinsic kinases Binding of G-CSF to its receptor causes dimerization or oligomerization of the receptor (Ishizaka-Ikeda et al., 1993). It is reported that at a low concentration of G-CSF, an asymmetric 2 : 1 receptor±ligand complex is formed, while at high ligand concentrations it is converted to 2 : 2 or 4 : 4 complex (Hiraoka et al., 1995; Horan et al., 1996). A 76 amino acid stretch proximal to the transmembrane domain, containing the box 1 and box 2 motifs, is essential for transducing growth signaling (Fukunaga et al.,

1948 Shigekazu Nagata 1993; Ziegler et al., 1993) while both the N- and Cterminal domains of the cytoplasmic region are indispensable for transducing differentiation signals (Dong et al., 1993; Fukunaga et al., 1993). The Cterminal domain of the cytoplasmic region of the mouse G-CSFR contains four tyrosine residues (Tyr703, Tyr728, Tyr743, and Tyr763), which are phosphorylated upon stimulation by G-CSF (Pan et al., 1993), and seems to be involved in different aspects of signal transduction (de Koning et al., 1996b; Yoshikawa et al., 1995). JAK1 is constitutively associated with the G-CSFR and becomes activated by the binding of G-CSF to the receptor (Nicholson et al., 1994). G-CSF also activates JAK2 and TYK2 (Shimoda et al., 1994; Tian et al., 1996; Avalos et al., 1997). The membrane-proximal region containing the box 1 and 2 motifs is required for the activation of the JAK kinases (Nicholson et al., 1995). In addition to the JAK family kinases, an srcrelated protein tyrosine kinase, Lyn, and a non-srcrelated Syk tyrosine kinase have been reported to be associated with the G-CSFR and activated upon GCSF stimulation (Corey et al., 1994). The requirement of Lyn kinase in the G-CSFR-mediated proliferation signal was demonstrated with a reconstitution system using a Lyn-deficient chicken B cell line (Corey et al., 1998). On the other hand, irradiated mice reconstituted with Syk-deficient fetal liver show no gross perturbations in G-CSF responsiveness, suggesting no requirement of Syk for G-CSF signaling (Turner et al., 1995). Several other tyrosine kinases such as Tec, a cytoplasmic src-related protein kinase, and p72sak tyrosine kinase, are tyrosine-phosphorylated and specifically activated by G-CSF (Matsuda et al., 1995; Miyazato et al., 1996). However, their physiological roles in G-CSF-induced signal transduction is unknown.

Cytoplasmic signaling cascades The JAK kinases strongly activate STAT3 and weakly activate STAT1, which leads to the formation of STAT1 and STAT3 homodimeric and heterodimeric complexes (Tian et al., 1994). Activation of other STAT proteins such as STAT5 and a novel STAT-like protein, STAT G in G-CSF-stimulated neutrophils has also been reported (Tweardy et al., 1995; Nicholson et al., 1996; Tian et al., 1996). The activation of STAT3, but not of either STAT1 or STAT5, requires the membrane-distal region of the G-CSFR which carries Tyr703 (de Koning et al., 1996b; Tian et al., 1996). The surrounding sequence of Tyr703 is YXXQ, which fits to the consensus sequence for the STAT3-docking site found in gp130

(Stahl et al., 1995). Although the tyrosine phosphorylation of the G-CSF receptor is not an absolute requirement for STAT3 activation (Cleveland et al., 1989; de Koning et al., 1996a; Nicholson et al., 1996; Welte et al., 1987), it is possible that its phosphorylation increases the affinity of STAT3 for this docking site. The activated, i.e. phosphorylated STAT3, seems to be released from the receptor by forming a homoor heterodimer with STAT1, and is translocated into the nucleus (Shimozaki et al., 1997). At high concentration of G-CSF, G-CSF activates STAT5 through the box 1 and box 2 region of G-CSFR, which is responsible for G-CSF-induced proliferation signals (Dong et al., 1998). The Ras/MAP kinase pathway is another signaling cascade activated by G-CSF. In the proB cell line BAF-B03, G-CSF activates Ras and MAP kinase (Bashey et al., 1994; Nicholson et al., 1995). Various molecules such as the Shc, Grb2, and Syp adaptors, and the vav guanine nucleotide exchanger are phosphorylated by G-CSF in various cells responding to G-CSF (de Koning et al., 1996b) , which are responsible for activation of the Ras/MAP kinase pathway. The activation of Ras requires the membrane-proximal region of G-CSFR (Barge et al., 1996), as well as the membrane-distal region, specifically the domain containing Tyr763 (Duronio et al., 1992; de Koning et al., 1996b). It is likely that the JAK family kinases, activated through the membrane-proximal region of the receptor, phosphorylate Tyr763 of the receptor, to which adaptor molecules are recruited to activate the Ras/MAP kinase pathway. The kinds of genes activated by the signal from the Ras/MAP kinases are not elucidated yet. In addition to tyrosine kinases and Ras/MAP kinase, G-CSF seems to regulate turnover of phosphatidylinositol. Upon binding of G-CSF to the receptor, phosphatidylinositol 3-kinase (PI-3 kinase) is recruited to the region containing Tyr-1 (amino acids 682±715), and it blocks apoptosis leading to cell survival (Hunter and Avalos, 1998). On the other hand, SH2-containing inositol phosphatase (SHIP) binds to the membrane-distal region of the receptor and, together with Shc, downregulates proliferation signals.

DOWNSTREAM GENE ACTIVATION

Transcription factors activated As described above, G-CSF activates STAT1 and STAT3. c-rel, a proto-oncogene belonging to the

G-CSF Receptor 1949 NFB family, was also shown to be activated by G-CSF (Druker et al., 1994; Avalos et al., 1995). Although the box 1 motif in the membrane-proximal region of the receptor is required for NFB activation, it is not clear what roles NFB plays in G-CSF-mediated signaling. One possibility is that NFB activation by G-CSF induces anti-apoptotic signals, as found in the IL-1 and TNF systems (Liu et al., 1996b).

Genes induced G-CSF induces in myeloid cells the expression of various neutrophil-specific genes such as myeloperoxidase (MPO) and neutrophilic elastase (Fukunaga et al., 1993; Morishita et al., 1987). Transcription of c-fos oncogene is upregulated in myeloid cells by treatment with G-CSF (Gonda and Metcalf, 1984), while the transcription of other oncogenes such as c-myc and c-myb is suppressed by the same treatment (Gonda and Metcalf, 1984; Shimozaki et al., 1997).

0

A DNA fragment of about 800 bp in the 5 flanking region of human myeloperoxidase gene responds to G-CSF for gene activation in a myeloid cell-specific manner (Suzow and Friedman, 1993; Orita et al., 1997). Several transcription factors, such as NF-Y, PEBP/CBP, and MyNF-1, were suggested to be involved in neutrophil-specific expression of myeloperoxidase (Suzow and Friedman, 1993; Nuchprayoon et al., 1994; Orita et al., 1997). However, the precise mechanism for the G-CSFinduced activation of neutrophil-specific genes is not elucidated yet.

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Unique biological effects of activating the receptors (production

of

Similar to the G-CSF-null mice, the mice lacking GCSFR show chronic neutropenia: the peripheral blood neutrophil level is 20±30% of those of wild-type mice (Liu et al., 1996a). The number of neutrophilic precursor cells is also reduced in G-CSFR-null mice, confirming an involvement of G-CSF in the initial stage of neutrophilic development. The residual neutrophils in G-CSFR-null mice rapidly undergo apoptosis.

Human abnormalities Severe congenital neutropenia (Kostmann's syndrome) is characterized by profound neutropenia and a maturation arrest of marrow progenitor cells at the promyelocyte±myelocyte stage. Somatic point mutations in one allele of the G-CSF receptor gene have been identified in some patients with severe congenital neutropenia (Dong et al., 1994, 1995, 1997).

References

Promoter regions involved

Granulopoiesis granulocytes).

Phenotypes of receptor knockouts and receptor overexpression mice

neutrophilic

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LICENSED PRODUCTS Mouse anti-human G-CSFR monoclonal antibody (clone LMM 741) (Layton et al., 1997a; Nicholson et al., 1994) is available from PharMingen (San Diego).