G-CSF Receptor Shigekazu Nagata* Department of Genetics, Osaka University Medical School, 2-2 Yamada-oka Suita, Osaka, 565-0871, Japan *corresponding author tel: (cid:135)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 G-CSFR is also known as CD114. which belongs to the cytokine receptor superfamily, and is specifically expressed in mature neutrophils and neutrophilic precursors. Binding of G-CSF to Structure G-CSFR induces its dimerization. The dimerized receptortransducesgrowthanddifferentiationsignals G-CSFR is a type I membrane protein of about whicharemediatedbytheN-terminalandC-terminal 130kDa. halves of the G-CSFR cytoplasmic region. G-CSF activates JAK family kinases, which cause tyrosine phosphorylation of STAT family transcription Main activities and factors. STATs then stimulate proliferation, and induce differentiation of neutrophilic precursor cells. pathophysiological roles Genetic defects in the G-CSFR gene cause severe neutropenia (Kostmann’s syndrome). G-CSFR transduces proliferation and differen- tiation signals into neutrophilic precursor cells, thus mediating the action of G-CSF (production BACKGROUND of neutrophils, granulopoiesis) (Fukunaga et al., 1991). Discovery The receptor for G-CSF was discovered as a GENE membrane protein expressed in myeloid leukemia cells or neutrophilic granulocytes to which [125I]- Accession numbers labeled G-CSF binds (Nicola and Metcalf, 1984). Murine G-CSFR was purified from the membrane Human G-CSFR: M59818 (Fukunaga et al., 1990c; fraction of mouse myeloid leukemia NFS60 cells that Larsen et al., 1990) respond to G-CSFfor proliferation (Fukunagaet al., Murine G-CSFR: M32699 (Fukunaga et al., 1990b), and its cDNA was isolated from an NFS60 1990a) cDNAlibrarybyexpressioncloning(Fukunagaetal., 1990a). The human counterpart was subsequently identified from a human placenta cDNA library by Sequence crosshybridization with mouse cDNA (Fukunaga et al., 1990c), or from a human neutrophil cDNA library by expression cloning (Larsen et al., 1990). See Figure 1. 1946 Shigekazu Nagata Figure 1 Nucleotide sequence for the human G-CSFR gene. Human G-CSFR 1 GAAGCTGGAC TGCAGCTGGT TTCAGGAACT TCTCTTGACG AGAAGAGAGA CCAAGGAGGC 61 CAAGCAGGGG CTGGGCCAGA GGTGCCAACA TGGGGAAACT GAGGCTCGGC TCGGAAAGGT 121 GAAGTAACTT GTCCAAGATC ACAAAGCTGG TGAACATCAA GTTGGTGCTA TGGCAAGGCT 181 GGGAAACTGC AGCCTGACTT GGGCTGCCCT GATCATCCTG CTGCTCCCCG GAAGTCTGGA 241 GGAGTGCGGG CACATCAGTG TCTCAGCCCC CATCGTCCAC CTGGGGGATC CCATCACAGC 301 CTCCTGCATC ATCAAGCAGA ACTGCAGCCA TCTGGACCCG GAGCCACAGA TTCTGTGGAG 361 ACTGGGAGCA GAGCTTCAGC CCGGGGGCAG GCAGCAGCGT CTGTCTGATG GGACCCAGGA 421 ATCTATCATC ACCCTGCCCC ACCTCAACCA CACTCAGGCC TTTCTCTCCT GCTGCCTGAA 481 CTGGGGCAAC AGCCTGCAGA TCCTGGACCA GGTTGAGCTG CGCGCAGGCT ACCCTCCAGC 541 CATACCCCAC AACCTCTCCT GCCTCATGAA CCTCACAACC AGCAGCCTCA TCTGCCAGTG 601 GGAGCCAGGA CCTGAGACCC ACCTACCCAC CAGCTTCACT CTGAAGAGTT TCAAGAGCCG 661 GGGCAACTGT CAGACCCAAG GGGACTCCAT CCTGGACTGC GTGCCCAAGG ACGGGCAGAG 721 CCACTGCTGC ATCCCACGCA AACACCTGCT GTTGTACCAG AATATGGGCA TCTGGGTGCA 781 GGCAGAGAAT GCGCTGGGGA CCAGCATGTC CCCACAACTG TGTCTTGATC CCATGGATGT 841 TGTGAAACTG GAGCCCCCCA TGCTGCGGAC CATGGACCCC AGCCCTGAAG CGGCCCCTCC 901 CCAGGCAGGC TGCCTACAGC TGTGCTGGGA GCCATGGCAG CCAGGCCTGC ACATAAATCA 961 GAAGTGTGAG CTGCGCCACA AGCCGCAGCG TGGAGAAGCC AGCTGGGCAC TGGTGGGCCC 1021 CCTCCCCTTG GAGGCCCTTC AGTATGAGCT CTGCGGGCTC CTCCCAGCCA CGGCCTACAC 1081 CCTGCAGATA CGCTGCATCC GCTGGCCCCT GCCTGGCCAC TGGAGCGACT GGAGCCCCAG 1141 CCTGGAGCTG AGAACTACCG AACGGGCCCC CACTGTCAGA CTGGACACAT GGTGGCGGCA 1201 GAGGCAGCTG GACCCCAGGA CAGTGCAGCT GTTCTGGAAG CCAGTGCCCC TGGAGGAAGA 1261 CAGCGGACGG ATCCAAGGTT ATGTGGTTTC TTGGAGACCC TCAGGCCAGG CTGGGGCCAT 1321 CCTGCCCCTC TGCAACACCA CAGAGCTCAG CTGCACCTTC CACCTGCCTT CAGAAGCCCA 1381 GGAGGTGGCC CTTGTGGCCT ATAACTCAGC CGGGACCTCT CGCCCCACCC CGGTGGTCTT 1441 CTCAGAAAGC AGAGGCCCAG CTCTGACCAG ACTCCATGCC ATGGCCCGAG ACCCTCACAG 1501 CCTCTGGGTA GGCTGGGAGC CCCCCAATCC ATGGCCTCAG GGCTATGTGA TTGAGTGGGG 1561 CCTGGGCCCC CCCAGCGCGA GCAATAGCAA CAAGACCTGG AGGATGGAAC AGAATGGGAG 1621 AGCCACGGGG TTTCTGCTGA AGGAGAACAT CAGGCCCTTT CAGCTCTATG AGATCATCGT 1681 GACTCCCTTG TACCAGGACA CCATGGGACC CTCCCAGCAT GTCTATGCCT ACTCTCAAGA 1741 AATGGCTCCC TCCCATGCCC CAGAGCTGCA TCTAAAGCAC ATTGGCAAGA CCTGGGCACA 1801 GCTGGAGTGG GTGCCTGAGC CCCCTGAGCT GGGGAAGAGC CCCCTTACCC ACTACACCAT 1861 CTTCTGGACC AACGCTCAGA ACCAGTCCTT CTCCGCCATC CTGAATGCCT CCTCCCGTGG 1921 CTTTGTCCTC CATGGCCTGG AGCCCGCCAG TCTGTATCAC ATCCACCTCA TGGCTGCCAG 1981 CCAGGCTGGG GCCACCAACA GTACAGTCCT CACCCTGATG ACCTTGACCC CAGAGGGGTC 2041 GGAGCTACAC ATCATCCTGG GCCTGTTCGG CCTCCTGCTG TTGCTCACCT GCCTCTGTGG 2101 AACTGCCTGG CTCTGTTGCA GCCCCAACAG GAAGAATCCC CTCTGGCCAA GTGTCCCAGA 2161 CCCAGCTCAC AGCAGCCTGG GCTCCTGGGT GCCCACAATC ATGGAGGAGG ATGCCTTCCA 2221 GCTGCCCGGC CTTGGCACGC CACCCATCAC CAAGCTCACA GTGCTGGAGG AGGATGAAAA 2281 GAAGCCGGTG CCCTGGGAGT CCCATAACAG CTCAGAGACC TGTGGCCTCC CCACTCTGGT 2341 CCAGACCTAT GTGCTCCAGG GGGACCCAAG AGCAGTTTCC ACCCAGCCCC AATCCCAGTC 2401 TGGCACCAGC GATCAGGTCC TTTATGGGCA GCTGCTGGGC AGCCCCACAA GCCCAGGGCC 2461 AGGGCACTAT CTCCGCTGTG ACTCCACTCA GCCCCTCTTG GCGGGCCTCA CCCCCAGCCC 2521 CAAGTCCTAT GAGAACCTCT GGTTCCAGGC CAGCCCCTTG GGGACCCTGG TAACCCCAGC 2581 CCCAAGCCAG GAGGACGACT GTGTCTTTGG GCCACTGCTC AACTTCCCCC TCCTGCAGGG 2641 GATCCGGGTC CATGGGATGG AGGCGCTGGG GAGCTTCTAG GGCTTCCTGG GGTTCCCTTC 2701 TTGGGCCTGC CTCTTAAAGG CCTGAGCTAG CTGGAGAAGA GGGGAGGGTC CATAAGCCCA 2761 TGACTAAAAA CTACCCCAGC CCAGGCTCTC ACCATCTCCA GTCACCAGCA TCTCCCTCTC 2821 CTCCCAATCT CCATAGGCTG GGCCTCCCAG GCGATCTGCA TACTTTAAGG ACCAGATCAT 2881 GCTCCATCCA GCCCCACCCA ATGGCCTTTT GTGCTTGTTT CCTATAACTT CAGTATTGTA 2941 AAC Chromosome location and linkages Mouse G-CSFR: P40223 (Fukunaga et al., 1990a) G-CSFR is on chromosome 1p35-34.3 in humans Sequence (Inazawa et al., 1991) and on chromosome 4 in the mouse (Ito et al., 1994). See Figure 2. PROTEIN Description of protein Accession numbers Human G-CSFR comprises 836 amino acids with a Human G-CSFR: Q99062 (Fukunaga et al., 1990c; signal sequence of 23 amino acids at the N-terminus Larsen et al., 1990) (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 W AALIILLLP G SLEE CGHIS VSAPIVHLGD PITASCIIKQ NCSHLDPEPQ ILWRLGAELQ PGGRQQRLSD GTQESIITLP HLNHTQAFLS CCLNWGNSLQ ILDQVELRAG YPPAIPHNLS CLMNLTTSSL ICQWEPGPET HLPTSFTLKS FKSRGNCQTQ GDSILDCVPK DGQSHCCIPR KHLLLYQNMG IWVQAENALG TSMSPQLCLD PMDVVKLEPP MLRTMDPSPE AAPPQAGCLQ LCWEPWQPGL HINQKCELRH KPQRGEASWA LVGPLPLEAL QYELCGLLPA TAYTLQIRCI RWPLPGHWSD WSPSLELRTT ERAPTVRLDT WWRQRQLDPR TVQLFWKPVP LEEDSGRIQG YVVSWRPSGQ AGAILPLCNT TELSCTFHLP SEAQEVALVA YNSAGTSRPT PVVFSESRGP ALTRLHAMAR DPHSLWVGWE PPNPWPQGYV IEWGLGPPSA SNSNKTWRME QNGRATGFLL KENIRPFQLY EIIVTPLYQD TMGPSQHVYA YSQEMAPSHA PELHLKHIGK TWAQLEWVPE PPELGKSPLT HYTIFWTNAQ NQSFSAILNA SSRGFVLHGL EPASLYHIHL MAASQAGATN STVLTLMTLT PEGSELH II L G LFGLLLLLT CLCGTAWLC C SPNRKNPLWP SVPDPAHSSL GSWVPTIMEE DAFQLPGLGT PPITKLTVLE EDEKKPVPWE SHNSSETCGL PTLVQTYVLQ GDPRAVSTQP QSQSGTSDQV LYGQLLGSPT SPGPGHYLRC DSTQPLLAGL TPSPKSYENL WFQASPLGTL VTPAPSQEDD CVFGPLLNFP LLQGIRVHGM EALGSF domain of 26 amino acids divides the molecule into Regulation of receptor expression the extracellular region of 604 amino acids, and the cytoplasmic region of 183 amino acids. Tworegionsinthepromoterseemtobeimportantfor G-CSF receptor gene expression. C/EBP(cid:11) binds to a Relevant homologies and species regionlocatedat(cid:255)49fromthetranscriptioninitiation differences site (Smith et al., 1996), which is essential for the expressionofG-CSFR(Zhangetal.,1997).Theother two cis-regulatory elements are located at (cid:135)36 and HumanandmouseG-CSFreceptorshaveahomology (cid:135)43, to which the ets family member PU.1. binds. of 62.5%, and there is no species specificity between Mutation of these sites reduces the promoter activity human and mouse G-CSFRs (Nicola et al., 1985; by 75% (Smith et al., 1996). However, mice lacking Fukunaga et al., 1990c). The overall structure of G- PU.1 can express G-CSFR (Olson et al., 1995), CSFR is similar to that of gp130 (the IL-6 receptor suggesting that this element may not be essential for signaltransducer)(Hibietal.,1990)andLIFreceptor the expression of the G-CSFR gene. (Gearing et al., 1991), and leptin receptor (Tartaglia etal.,1995).Theligand-bindingdomain(CRHdomain, aminoacidsfrom97to308inmouseG-CSFR)inthe Release of soluble receptors extracellularregionofG-CSFR(Fukunagaetal.,1991; Layton et al., 1997b) shows similarity to the corre- An alternatively spliced mRNA coding for a protein sponding region of other cytokine receptors (Bazan, lacking the transmembrane region can be detected in 1990a,b;Fukunagaetal.,1990a,c).Tworegions(box1 humanU937cells(Fukunagaetal.,1990c).However, and box 2) of the membrane-proximal region of theexistenceofsolubleG-CSFRinhumanserumhas G-CSFR are related to the corresponding regions of not yet been reported. othercytokinereceptors,whileanotherregion(box3) is related to the corresponding region of gp130 (Fukunagaetal.,1991;Murakamietal.,1991). SIGNAL TRANSDUCTION Affinity for ligand(s) Associated or intrinsic kinases G-CSF binds to G-CSFR with K of about 100pM d Binding of G-CSF to its receptor causes dimerization (Fukunaga et al., 1990b; Nicola and Metcalf, 1984). or oligomerization of the receptor (Ishizaka-Ikeda Cell types and tissues expressing etal.,1993).Itisreportedthatatalowconcentration of G-CSF, an asymmetric 2:1 receptor–ligand the receptor complex is formed, while at high ligand concentra- tions it is converted to 2:2 or 4:4 complex (Hiraoka G-CSFR is expressed in neutrophils, neutrophilic et al., 1995; Horan et al., 1996). A 76 amino acid precursors in the bone marrow, myeloid leukemia stretch proximal to the transmembrane domain, cells, and placenta (Fukunaga et al., 1990a,c; Nicola containing the box 1 and box 2 motifs, is essential and Metcalf, 1984). for transducing growth signaling (Fukunaga et al., 1948 Shigekazu Nagata 1993; Ziegler et al., 1993) while both the N- and C- (Stahl et al., 1995). Although the tyrosine phosphor- terminal domains of the cytoplasmic region are ylation of the G-CSF receptor is not an absolute indispensable for transducing differentiation signals requirement for STAT3 activation (Cleveland et al., (Dong et al., 1993; Fukunaga et al., 1993). The C- 1989; de Koning et al., 1996a; Nicholson et al., 1996; terminal domain of the cytoplasmic region of the Welte et al., 1987), it is possible that its phosphoryla- mouse G-CSFR contains four tyrosine residues tion increases the affinity of STAT3 for this docking (Tyr703, Tyr728, Tyr743, and Tyr763), which are site.Theactivated,i.e.phosphorylatedSTAT3,seems phosphorylated upon stimulation by G-CSF (Pan to be released from the receptor by forming a homo- et al., 1993), and seems to be involved in different or heterodimer with STAT1, and is translocated into aspects of signal transduction (de Koning et al., the nucleus (Shimozaki et al., 1997). At high 1996b;Yoshikawaetal.,1995).JAK1isconstitutively concentration of G-CSF, G-CSF activates STAT5 associated with the G-CSFR and becomes activated through the box 1 and box 2 region of G-CSFR, by the binding of G-CSF to the receptor (Nicholson which is responsible for G-CSF-induced proliferation et al., 1994). G-CSF also activates JAK2 and TYK2 signals (Dong et al., 1998). (Shimoda et al., 1994; Tian et al., 1996; Avalos et al., The Ras/MAP kinase pathway is another signaling 1997).Themembrane-proximalregioncontainingthe cascade activated by G-CSF. In the proB cell line box1and2motifsisrequiredfortheactivationofthe BAF-B03, G-CSF activates Ras and MAP kinase JAK kinases (Nicholson et al., 1995). (Bashey et al., 1994; Nicholson et al., 1995). Various In addition to the JAK family kinases, an src- molecules such as the Shc, Grb2, and Syp adaptors, related protein tyrosine kinase, Lyn, and a non-src- and the vav guanine nucleotide exchanger are phos- related Syk tyrosine kinase have been reported to be phorylated by G-CSF in various cells responding to associated with the G-CSFR and activated upon G- G-CSF(deKoningetal.,1996b),whichareresponsible CSFstimulation(Coreyetal.,1994).Therequirement for activation of the Ras/MAP kinase pathway. The of Lyn kinase in the G-CSFR-mediated proliferation activation of Ras requires the membrane-proximal signal was demonstrated with a reconstitution system region of G-CSFR (Barge et al., 1996), as well as the using aLyn-deficient chickenBcell line (Corey et al., membrane-distal region, specifically the domain 1998). On the other hand, irradiated mice reconsti- containing Tyr763 (Duronio et al., 1992; de Koning tuted with Syk-deficient fetal liver show no gross etal.,1996b).Itis likelythatthe JAKfamilykinases, perturbations in G-CSF responsiveness, suggesting activated through the membrane-proximal region of no requirement of Syk for G-CSF signaling (Turner thereceptor,phosphorylateTyr763ofthereceptor,to et al., 1995). Several other tyrosine kinases such as which adaptor molecules are recruited to activate the Tec, a cytoplasmic src-related protein kinase, and Ras/MAP kinase pathway. The kinds of genes p72sak tyrosine kinase, are tyrosine-phosphorylated activated by the signal from the Ras/MAP kinases and specifically activated by G-CSF (Matsuda et al., are notelucidated yet. 1995; Miyazato et al., 1996). However, their physiol- In addition to tyrosine kinases and Ras/MAP ogical roles in G-CSF-induced signal transduction is kinase, G-CSF seems to regulate turnover of phos- unknown. phatidylinositol. Upon binding of G-CSF to the receptor, phosphatidylinositol 3-kinase (PI-3 kinase) is recruited to the region containing Tyr-1 (amino Cytoplasmic signaling cascades acids682–715),anditblocksapoptosisleadingtocell survival (Hunter and Avalos, 1998). On the other The JAK kinases strongly activate STAT3 and hand, SH2-containing inositol phosphatase (SHIP) weaklyactivateSTAT1,whichleadstotheformation binds to the membrane-distal region of the receptor of STAT1 and STAT3 homodimeric and hetero- and, together with Shc, downregulates proliferation dimeric complexes (Tian et al., 1994). Activation of signals. 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., DOWNSTREAM GENE 1995; Nicholson et al., 1996; Tian et al., 1996). The ACTIVATION activation of STAT3, but not of either STAT1 or STAT5, requires the membrane-distal region of the Transcription factors activated 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 As described above, G-CSF activates STAT1 and sequence for the STAT3-docking site found in gp130 STAT3. c-rel, a proto-oncogene belonging to the G-CSF Receptor 1949 NF(cid:20)B family, was also shown to be activated by Phenotypes of receptor knockouts G-CSF (Druker et al., 1994; Avalos et al., 1995). and receptor overexpression mice Although the box 1 motif in the membrane-proximal region of the receptor is required for NF(cid:20)B activation, it is not clear what roles NF(cid:20)B plays in Similar to the G-CSF-null mice, the mice lacking G- G-CSF-mediated signaling. One possibility is that CSFRshowchronicneutropenia:theperipheralblood NF(cid:20)B activation by G-CSF induces anti-apoptotic neutrophil level is 20–30% of those of wild-type mice signals, as found in the IL-1 and TNF systems (Liu (Liu et al., 1996a). The number of neutrophilic et al., 1996b). precursor cells is also reduced in G-CSFR-null mice, confirminganinvolvementofG-CSFintheinitialstage ofneutrophilicdevelopment.Theresidualneutrophils inG-CSFR-nullmicerapidlyundergoapoptosis. Genes induced Human abnormalities G-CSF induces in myeloid cells the expression of various neutrophil-specific genes such as myeloper- oxidase (MPO) and neutrophilic elastase (Fukunaga Severe congenital neutropenia (Kostmann’s syndrome) et al., 1993; Morishita et al., 1987). Transcription of is characterized by profound neutropenia and a c-fos oncogene is upregulated in myeloid cells by maturation arrest of marrow progenitor cells at the treatment with G-CSF (Gonda and Metcalf, 1984), promyelocyte–myelocyte stage. Somatic point muta- while the transcription of other oncogenes such as tions in one allele of the G-CSF receptor gene have c-myc and c-myb is suppressed by the same beenidentifiedinsomepatientswithseverecongenital treatment (Gonda and Metcalf, 1984; Shimozaki neutropenia (Dong et al., 1994, 1995, 1997). et al., 1997). References Promoter regions involved Avalos, B. R., Hunter, M. G., Parker, J. M., Ceselski, S. K., Druker, B. J., Corey, S. J., and Mehta, V. B. (1995). Point A DNA fragment of about 800bp in the 50 mutations in the conserved box 1 region inactivate the human flanking region of human myeloperoxidase gene granulocyte colony-stimulating factor receptor for growth sig- responds to G-CSF for gene activation in a myeloid nal transduction and tyrosine phosphorylation of p75c-rel. cell-specific manner (Suzow and Friedman, 1993; Blood8535,3117–3126. Orita et al., 1997). Several transcription factors, such Avalos, B. R., Parker, J. M., Ware, D. A., Hunter, M. G., Sibert, K. 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