Hematopoietic Receptor Family

Hematopoietic Receptor Family Atsushi Miyajima* Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Tokyo, Bunkyo-Ku, 113-0032, Japan * corresponding author tel: +81-3-5800-3551, fax: +81-3-4800-3550, e-mail: [email protected] DOI: 10.1006/rwcy.2000.02010.

SUMMARY The functional high-affinity receptors for IL-3, IL-5, and GM-CSF are heterodimeric receptors consisting of a cytokine-specific  subunit and the common subunit, c, which is shared by the three cytokines. In the mouse, the IL-3-specific IL3 subunit, homologous to c, is present and forms a high-affinity receptor only with the IL-3R. These subunits are members of the type I cytokine receptor family. As the subunits play a major role in signaling, the three cytokines exhibit similar biological functions on the same target cells. The JAK2 tyrosine kinase is associated with both subunits and is activated upon cytokine stimulation. The membrane-proximal region of c is responsible for the activation of JAK2 and STAT5 as well as induction of c-myc. The signals induced by this region are required for cell cycle progression and DNA synthesis. The activation of the Ras pathway requires the distal region of c and is involved in suppression of apoptosis. As IL-3, IL-5, and GM-CSF are produced mainly from activated T cells and mast cells, it has been hypothesized that these cytokines play a major role in inductive hematopoiesis associated with immune and inflammatory reactions. However, mice lacking the entire functions of IL-3, IL-5, and GM-CSF are viable and no significant defects other than a reduced level of eosinophils and some defects in mast cell development were found, even in mice infected with bacteria and parasites. Thus, there appears to be a compensatory

mechanism to overcome any defect in the three cytokines.

INTRODUCTION IL-3, IL-5, and GM-CSF are related to each other genetically, structurally, and functionally, and stimulate development of hematopoietic cells. IL-3 and GM-CSF act on a broad range of hematopoietic progenitors and thereby exhibit a number of different biological activities, whereas IL-5-responsive cells are restricted to eosinophils, basophils, and some mouse B cells. IL-3, GM-CSF, and IL-5 exhibit almost identical biological activities when they act on the same target cells such as eosinophilic progenitors. These cytokines are mainly produced by activated T cells and mast cells, but the production by bone marrow stroma cells is minimal. Thus, it has been hypothesized that these cytokines play a major role in hematopoiesis in an emergency situation such as inflammation (Arai et al., 1990). The common activities of the three cytokines are now explained by the structures of the receptors. The receptors for IL-3, IL-5, and GM-CSF are composed of a cytokine-specific  subunit and a common subunit, c (Miyajima et al., 1993). As the c subunit plays a critical role in signal transduction, similar signals are transmitted through the subunit, and the  subunits provide specificity to cytokines. This chapter describes the structure, expression, and functions of the IL-3, IL-5, and GM-CSF receptors.

1892 Atsushi Miyajima

STRUCTURE OF THE IL-3, IL-5, AND GM-CSF RECEPTORS The high-affinity receptors for IL-3, GM-CSF, and IL-5 consist of a cytokine-specific  subunit and a common subunit, c (Miyajima et al., 1993) (Figure 1). Both are members of the class I cytokine receptor superfamily. The  subunits are glycoproteins of 60± 70 kDa with a small cytoplasmic domain of about 50 amino acid residues and bind their specific ligand with low affinity. The shared c is a glycoprotein of 120±130 kDa with two repeats of a conserved motif of the class I cytokine receptors in the extracellular domain. Its large cytoplasmic domain contains two motifs known as Box-1 and Box-2 which are conserved in members of the class I cytokine receptors. The c does not bind any cytokine by itself, but is required for the formation of a high-affinity receptor with any of the three  subunits, IL-3 receptor  (IL-3R), GM-CSF receptor  (GM-CSFR), and IL-5 receptor  (IL-5R). IL-3R and GM-CSFR

genes are colocalized in the human pseudoautosomal region of the sex chromosomes (Gough et al., 1990), whereas the IL-5R gene is on human chromosome 3p25±p26 (Isobe et al., 1992). In the mouse, there are two homologous subunits: c (also known as AIC2B) and the IL-3-specific subunit, IL3 (also known as AIC2A) (Itoh et al., 1990; Gorman et al., 1990), which binds IL-3 with low affinity and forms a high-affinity receptor with IL-3R only (Hara and Miyajima, 1992) (Figure 1). No functional differences have been found between the two high-affinity mIL-3Rs which consist of either c or IL3. The two subunit genes are tightly linked on mouse chromosome 15 (Gorman et al., 1992) and are placed in a head-to-head configuration (Hannemann et al., 1995). The human c gene is on chromosome 22q12.2±13.1 (Shen et al., 1992) and there is no evidence that IL3 exists in the human. Like receptor tyrosine kinases, multimerization of the receptor subunits is a key step for activation of the class I cytokine receptors. Although the cytoplasmic domains of both  and c are required

Figure 1 Receptors for IL-3, GM-CSF, and IL-5. The high-affinity receptors for IL-3, GM-CSF, and IL-5 consist of a cytokine-specific  subunit and the common subunit, c. There is the IL-3-specific subunit IL3 in the mouse but not in the human.

mIL-3R

IL3Rα

IL-3R

IL3Rα

βIL3

GM-CSFR IL-5R

GMRα

βc

IL5Rα

βc

βc

Hematopoietic Receptor Family 1893 for signaling, the c cytoplasmic domain plays a major role in signal transduction. This is supported by experiments using chimeric receptors that are forced to form a dimer, e.g. the chimeric receptor which consists of the c cytoplasmic domain and the extracellular domain of homodimerizing EPOR induced full growth signals in response to EPO (Sakamaki et al., 1993), whereas similar chimeric receptors with a cytoplasmic domain of the  subunits failed to induce any signals (Eder et al., 1994; Muto et al., 1995). Thus the  subunits provide the cytokine specificity and c induces common signals. This model clearly explains the functional overlap between the three cytokines. Alanine substitution mutagenesis of c identified the amino acid residues of c (Y365±I368) essential for binding to GM-CSF (Woodcock et al., 1994). Evidence was presented that the GM-CSF receptor exists as a preformed complex that can be activated by GM-CSF, IL-3, and IL-5 (Woodcock et al., 1997) and the IL-3 and GM-CSF receptors undergo covalent dimerization of the respective  subunit with c in the presence of cognate cytokine. Cys86, Cys91, and Cys96 of c are involved in the covalent dimerization (Stomski et al., 1998). IL-3 was shown to induce disulfide-linked dimerization between IL-3R and c, which is required for receptor activation but not for high-affinity binding (Stomski et al., 1996). It was also reported that c forms an inactive dimer in the absence of cognate ligand and is activated by binding of a cytokine (Muto et al., 1996). These results suggest that the activated receptor complex may be a multimeric complex. Polymerase chain reaction (PCR)-based random mutagenesis of the c subunit led to identification of several mutations that result in constitutive dimerization of c (Jenkins et al., 1995; Jenkins et al., 1998). Mutations in the extracellular domain of c are clustered in the membrane-proximal domain (domain 4). Two mutations in the transmembrane domain were found and one of them (V449E) is similar to the constitutive active mutant of the Neu/ ErbB oncogene. Interestingly, mutations at two positions (R461C,H and H544R) in the cytoplasmic domain also result in constitutive activity. Whether these constitutive active c form either a dimer or a more complex structure remains unknown.

EXPRESSION OF THE RECEPTOR SUBUNITS Expression of  and subunits of the IL-3/GM-CSF/ IL-5 receptors is mainly restricted to hematopoietic cells (Sato et al., 1993a), while some expression is

also found in nonhematopoietic tissues such as testis, placenta, and brain (Morikawa et al., 1996; Korpelainen et al., 1993; Gearing et al., 1989). c and IL3 are expressed in various myeloid progenitor cells, macrophages, mast cells, CD5-positive B cells, and some endothelial cells, but not in erythroblasts, mature T cells, and fibroblasts (Gorman et al., 1990). Expression of the  subunits is more restricted to cytokine-responsive cells: IL-3R, but not GMCSFR and IL-5R, is expressed in mast cells and multipotential progenitor cells that form the CFU mix. In contrast, IL-5R is predominantly expressed in eosinophils and a subset of B cells, and IL-5 is a major cytokine for eosinophils, but not for other hematopoietic cells (Takatsu et al., 1994). IL-3 and GM-CSF exhibit a broad spectrum of biological functions, while IL-5 function is restricted to mainly eosinophils (Arai et al., 1990). The functional differences between the three cytokines may be due to the restricted expression of the  subunits. Alternatively, each  subunit may play an active role in inducing cytokine-specific functions. These possibilities were tested by generating a transgenic mouse strain that expresses IL-5R ubiquitously by the constitutive promoter of the phosphoglycerokinase 1 (PGK) gene (Takagi et al., 1995). Bone marrow cells of the transgenic mice formed colonies of various lineages and mixed colonies in response to IL-5 in a manner similar to IL-3, indicating that the limited activity of IL-5 is mainly due to the restricted expression of IL-5R and that IL-5R is functionally equivalent to IL-3R. The results further suggest that hematopoietic cells have their own differentiation program which is not affected by the  subunits. However, it should be noted that the cytoplasmic domains of the  subunits are required for signaling.

MICE DEVOID OF THE IL-3, GM-CSF, AND IL-5 RECEPTORS As there are two IL-3 receptors in the mouse (Miyajima et al., 1993), either IL3 or c may be dispensable for IL-3 function, whereas c is crucial for IL-5 and GM-CSF. To test the role of each subunit, mice devoid of either one of the subunits were generated (Nishinakamura et al., 1995). As expected, the IL3-deficient mice showed no apparent phenotype and no hematological defect was found. In contrast, bone marrow cells of the c-deficient mice did not form any colonies in the presence of either GMCSF or IL-5, while IL-3-induced colony formation was normal, indicating that c is essential for the function of GM-CSF and IL-5. A significant reduction of the

1894 Atsushi Miyajima number of eosinophils in the peripheral blood was noticed in the c-deficient mouse, consistent with the idea that IL-5 is the major cytokine for eosinophil development (Takatsu et al., 1994). This is consistent with the observation that IL-5R knockout mice exhibited only a basal level of eosinophils. In addition, IL-5R knockout mice showed decreased numbers of B-1 cells concomitant with low serum concentrations of IgM and IgG3 (Yoshida et al., 1996). The c-deficient mice exhibited lung abnormalities, including accumulation of proteinous material in the alveolar spaces and peribronchovascular lymphocytic infiltration. These observations are probably attributable to the deficiency of GM-CSF function as the same phenotype was observed in the GM-CSF-deficient mice (Dranoff et al., 1994). Alveolar macrophages may play a role in lung homeostasis by clearing surfactant and other debris from the alveolar space and the function of these macrophages may be impaired in such mice. The apparently normal hematopoiesis in the cdeficient mice may be due to the presence of the functional IL-3 receptor consisting of IL3. As IL-3 knockout mice were generated and were apparently normal, the IL-3 ligand knockout mouse was crossed with the c knockout mouse to generate a mouse line lacking the entire function of IL-3, GM-CSF, and IL-5 (Nishinakamura et al., 1996). Interestingly, the mice developed normally and were fertile. Hematopoiesis in these mice was similar to the c knockout. While the eosinophil number was reduced and lung disease developed in the double knockout mice, the severity was not changed compared to that of c mutant mice. Thus, the entire function of IL-3, GMCSF, and IL-5 is dispensable for hematopoiesis in normal life. Since a major source of these cytokines is activated T cells and basal levels of these cytokines are almost negligible in the normal bone marrow, it is possible that the major role of these cytokines is to promote hematopoiesis in an emergency situation such as inflammation (Arai et al., 1990). To address the question whether these cytokines are essential for emergency hematopoiesis, the mutant mice were infected with parasites and bacteria. IL-5R knockout mice showed sensitivity to infection with Angiostrongylus cantonensis (Sugaya et al., 1997). While IL-3 was shown not to be required for the generation of mast cells and basophils, it was recently shown to contribute to increases in the numbers of tissue mast cells, enhanced basophil production and immunity in mice infected with the nematode Stronglyoides venezuelensis (Lantz et al., 1998). However, the susceptibility to infection appears to be rather restricted to particular cases, and

the double knockout mice lacking both c and IL-3 were completely resistant to infection by Listeria monocytogenes (Nishinakamura et al., 1996). Thus, it appears that IL-3, GM-CSF, and IL-5 are largely dispensable even in emergency situations and the defects can be compensated by other unknown mechanism. Consistent with the notion that IL-3 is dispensable, naturally occurring mice with IL-3 hyporesponsiveness were found. It was known that several mouse strains such as A/J show hyporesponsiveness to IL-3. Molecular genetic analysis revealed that the defect is due to a small deletion (four base pairs) in the branch point in intron 7 of the IL-3R gene. As branch points are required for proper splicing, the A/J mouse produces mostly aberrant IL-3R mRNA (Ichihara et al., 1995). Curiously, the same mutation was found in 10 out of 27 laboratory mouse strains we analyzed. These mouse strains include A/J, C58J, A/WySnJ, A/HeY, RF/J, AKR/J, SM/J, BUB/BnJ, CE/J, and NZB/BINJ (Hara et al., 1995).

SIGNAL TRANSDUCTION IL-3, GM-CSF, and IL-5 induce rapid tyrosine phosphorylation of various cellular proteins including the subunit, phosphatidylinositol 3-kinase, Vav, Shc, and PTP-1D (Miyajima et al., 1993; Itoh et al., 1996). While members of the Src family of tyrosine kinases, such as Lyn and Fyn, as well as Btk, a member of another tyrosine kinase family, were initially shown to be involved in signaling in certain cell types (Torigoe et al., 1992; Li et al., 1995), the roles of these kinases in IL-3/GM-CSF signaling still remain unknown. In contrast, JAK kinases are now believed to play a major role in cytokine signaling (Ihle, 1995), and JAK2 was found to bind to the subunits. The conserved motif among various cytokine receptors known as Box-1 is present in the membrane-proximal region of the c subunit (between 455 and 544) and is sufficient for activation of JAK2 (Quelle et al., 1994), followed by activation of signal transducer and activator of transcription 5 (STAT5) (Figure 2). STAT5 activation leads to the induction of various genes such as Pim-1, Id-1, CIS, and OSM (Yoshimura et al., 1995, 1996; Mui et al., 1996). This region is also responsible for induction of several cytokineinducible genes, including c-myc. The signals derived from the membrane-proximal region are important for DNA replication and cell cycle progression. The more distal portion (544±626) of c is required for activation of Ras, Raf, MAP kinase and PI-3 kinase as well as induction of c-fos and c-jun (Sato et al., 1993b; Itoh et al., 1996). Analysis of signaling

Hematopoietic Receptor Family 1895 Figure 2 Signal transduction pathways from the IL-3/IL-5/GM-CSF receptors. JAK2 kinase is associated with the subunit and is activated by cytokine binding. The activated JAK2 induces various signals. Ras activated by SOS in turn activates the Raf/MAP kinase pathway and also PI-3 kinase. PI-3 kinase can be activated directly by the receptor as well. The activated STAT5 translocates to the nucleus and induces various genes, including CIS, a negative feedback regulator which turns off the STAT5 pathway. STAM1 appears to link JAK2 and induction of c-myc. Cell proliferation requires signals for suppression of cell death and for cell cycle progression; antiapoptotic signals are mainly delivered by the Ras pathway and signals for cell cycle progression are derived from the membraneproximal region of the subunit.

Cytokine

Jak2 G r b Shc Sos PTPase

STAT5 STAT5

STAM

Ras

CIS

Raf PI3K MAPKK c-Akt MAPK

c-Fos etc.

?

CIS, OSM Id, Pim,etc.

Anti-apoptosis

c-Myc

Cell Cycle Progression

Growth

1896 Atsushi Miyajima by mutant subunits with a C-terminal truncation as well as substitution mutants in which tyrosine residues are substituted with phenylalanine within the cytoplasmic domain suggests that multiple regions mediate Ras activation (Sato et al., 1993b; Itoh et al., 1996): Shc is an adapter molecule that is bound to the tyrosine phosphorylated receptor and recruits SOS, a Rasguanine nucleotide exchange factor, to membranes where Ras activation takes place. Phosphorylation of Shc by GM-CSF requires the tyrosine residue 577 of c. A protein tyrosine phosphatase, PTP-1D, is also known to serve as an adapter to recruit SOS, and its tyrosine phosphorylation is mediated by Tyr577 as well as other tyrosine residues downstream (Itoh et al., 1996). Thus, activation of Ras appears to be mediated by multiple pathways. Activation of Ras results in suppression of apoptosis of hematopoietic cells (Kinoshita et al., 1995). While Ras activates Raf kinase as well as PI-3 kinase, both pathways are involved in suppression of apoptosis (Kinoshita et al., 1997). Long-term cell proliferation requires signals from the membrane-proximal region of the subunit as well as the antiapoptotic function mediated by Ras (Kinoshita et al., 1995). A further C-terminal portion (763 to the C-terminus) appears to be involved in negative regulation, as the deletion of this region rather enhances signaling such as tyrosine phosphorylation of the subunit and Shc (Sato et al., 1993b). A tyrosine phosphatase may bind to this region and negatively regulate the signaling. The best candidate of this negative regulator is PTP1C (HCP) as it has been shown to bind to the subunit and is also known to be a negative regulator of cytokine signaling (Yi et al., 1993).

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