Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 947
_____________________________ _____________________________
Specific Signaling Through Heteromeric PDGF
Receptor Complexes
BY
SIMON EKMAN
ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2000
Dissertation for the Degree of Doctor of Medical Science in Molecular Cell Biology presented at Uppsala University in 2000
ABSTRACT
Ekman, S. 2000. Specific signaling through heteromeric PDGF receptor complexes. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 947. 80pp. Uppsala. ISBN 91-554-4785-6
Platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant for mesenchymal cells and exert its effect by binding to two structurally related receptor tyrosine kinases, denoted α- and β-receptors. PDGF binding induces dimerization of its receptors, both homo-and heterodimerization, leading to their autophosphorylation on tyrosine residues and binding of downstream signaling molecules. This thesis describes autophosphorylation and binding of signal transduction molecules to homo- and heterodimeric PDGF receptor complexes.
Heterodimeric PDGF receptor complexes have been found to mediate a stronger mitogenic response than homodimeric receptor complexes. It was found that Tyr771 in the PDGF β-receptor was significantly less phosphorylated in the heterodimeric β-receptor compared to the homodimeric receptor, and this correlated with reduced binding of GTPase activating protein (GAP) for Ras and decreased activation of the Ras/Mitogen activated protein kinase pathway.
The mechanism behind the lowered phosphorylation of Tyr771 in the heterodimeric PDGF β-receptor was investigated. It was found that the SH2 domain-containing tyrosine phosphatase SHP-2 was responsible, at least in part, for the dephosphorylation of Tyr771 in the heterodimeric β-receptor.
PDGF-induced autophosphorylation of tyrosine residues in the receptors has been proposed to occur in trans between the receptor molecules in the dimers. We demonstrated by phosphopeptide mapping that all major autophosphorylation sites can be phosphorylated in trans, both in the PDGF α- and β-receptors. Analyses of the abilities of heterodimeric receptor complexes of one kinase-active and one kinase-inactive receptor to mediate mitogenicity, chemotaxis and activation of mitogen activated protein kinase revealed that the signaling capacities were retained. This illustrates a functional co-operation between the two receptor molecules in the dimer, where one receptor provides a functional kinase and the other acts as a substrate and provides docking sites for downstream signaling molecules.
Elucidating the mechanisms behind the unique signaling properties of the heterodimeric PDGF receptor complex, two heterodimer-specific autophosphorylation sites, Tyr692 and Tyr970, were identified and found to interact with the low molecular weight protein tyrosine phosphatase (LMW-PTP). Mutation of Tyr692 or Tyr970 to phenylalanine residues did not affect PDGF-induced mitogenicity, but the Tyr692 to phenylalanine mutation reduced the chemotactic response mediated by the heterodimeric PDGF receptor complex. A mechanism for the lowered chemotactic response was found to involve an increased RasGAP binding to the heterodimeric β-receptor.
Simon Ekman, Ludwig Institute for Cancer Research, Biomedical Center, Box 595, S-751 24 Uppsala, Sweden
Simon Ekman 2000 ISSN 0282-7476 ISBN 91-554-4785-6
Printed in Sweden by Eklundshofs Grafiska, Uppsala 2000
To
My Wife
Lena
This thesis is based on the following papers, referred to in the text by their by their Roman numerals:
I: Ekman, S., Rupp Thuresson, E., Heldin, C.-H. and Rönnstrand, R. (1999). Increased mitogenicity of an αβ heterodimeric PDGF receptor complex correlates with lack of RasGAP binding. Oncogene. 18, 2481-2488
II: Emaduddin, M., Ekman, S., Rönnstrand, L. and Heldin, C.-H. (1999). Functional co- operation between the subunits in heterodimeric platelet-derived growth factor receptor complexes. Biochem. J. 341, 523-528.
III: Ekman, S., Hellman, U., Rönnstrand, L. and Heldin, C.-H. (2000). Involvement of LMW-PTP in heterodimeric PDGF receptor signaling for chemotaxis. Manuscript.
IV: Ekman, S., Engström, U., Heldin, C.-H. and Rönnstrand, L. (2000). SHP-2 is involved in heterodimer specific loss of phosphorylation of Tyr771 in the PDGF β-receptor. Manuscript.
En problemlösare värd namnet måste vara begåvad med två oförenliga egenskaper – rastlös fantasi och tålmodig envishet.
Howard W. Eves
Reprints were made with permission from the publishers
Paper II: Reproduced with permission from Emaduddin, M., Ekman, S., Rönnstrand, L. and Heldin, C.-H. (1999) Biochem. J. 341, 523-528. Biochemical Society
TABLE OF CONTENTS
Abbreviations Introduction
Platelet-derived growth factor and its receptors
Platelet-derived growth factor
Receptors for platelet-derived growth factor
Functions of PDGF and PDGF receptors in vivo
Cellular effects of PDGF Cell proliferation Cell motility Other effects
Physiological role of PDGF and PDGF receptors Embryonic development
Wound healing PDGF in diseases
Malignancies Atherosclerosis Fibrosis
Inflammation
Signal transduction via PDGF receptors
Receptor activation
Protein modules in signal transduction molecules Signaling pathways utilized by PDGF receptors
The Ras/MAP kinase pathway Src family kinases
Phosphatidylinositol 3´-kinase (PI3-K) Phospholipase C-γ
Protein tyrosine phosphatases - SHP-2
- LMW-PTP Crk
Other signaling molecules downstream of the PDGF receptors Signal diversification by homo- and heterodimerization
Present investigations
Mechanism for the increased mitogenicity of an αβ heterodimeric PDGF receptor complex (papers I and IV)
Functional co-operation between the subunits in heterodimeric platelet-derived growth factor receptor complexes (paper II)
Involvement of LMW-PTP in heterodimeric PDGF receptor signaling for chemotaxis (paper III)
Future perspectives
Acknowledgements
References
Abbreviations
CSF-1 Colony stimulating factor-1
Csw Corkscrew
DAG Diacylglycerol
Dok Downstream of tyrosine kinases
ECM Extracellular matrix
EGF Epidermal growth factor
Erk Extracellular regulated kinase
FAK Focal adhesion kinase
FGF Fibroblast growth factor
Flk-2 Fetal liver kinase-2
GAP GTPase activating protein
GDP Guanosine disphosphate
GEF Guanine nucleotide exchange factor
GTP Guanosine trisphosphate
IRK Insulin receptor kinase
Jak Janus kinase
JNK Jun N-terminal kinase
KSR-1 Kinase suppressor of Ras-1
LMW-PTP Low molecular weight protein tyrosine phosphatase
MAP Mitogen activated protein
MEK MAPK/ERK kinase
NF1 Neurofibromin 1
NGF Nerve growth factor
PAE Porcine aortic endothelial
PDGF Platelet-derived growth factor
PDZ PSD-95, DlgA, ZO-1
PH Pleckstrin homology
PI3-K Phosphatidylinositol 3´-kinase
PKB Protein kinase B
PKC Protein kinase C
PLC-γ Phospholipase C-γ
PRLr Prolactin receptor
PTB Phosphotyrosine binding
PtdIns Phosphatidylinositol
PtdIns(4,5)P2 Phosphatidylinositol (4,5) bisphosphate PtdIns(3,4,5)P3 Phosphatidylinositol (3,4,5) trisphosphate
PTK Protein tyrosine kinase
PTP Protein tyrosine phosphatase
SCF Stem cell factor
SH Src homology
SHP-2 Src homology containing phosphatase-2
Sos Son of sevenless
Stat Signal transducer and activator of transcription
TGF-β Transforming growth factor-beta
VEGF Vascular endothelial growth factor
Introduction
Life of a multicellular organism is dependent on communication between the cells within the organism. To maintain physiologically correct conditions, there is a need for a strict control of various cell activities such as proliferation, migration, differentiation and cell death. To orchestrate and ensure a proper balance between these activities, different signal transduction networks have evolved. For the communication outside the cells, soluble signaling molecules and extracellular matrix (ECM) components deliver the signals between and to the cells. On the cell surface, there are membrane spanning proteins that receive the signals from outside the cell and propagate them through the cell membrane and into the cell. These proteins traversing the cell membrane represent many different types of proteins differing in structure and enzymatic activities. They include receptors with tyrosine kinase or serine/threonine kinase activities, cytokine receptors, G-protein coupled receptors, antigen receptors, integrins and receptor-like protein phosphatases. Inside the cell, the signals are transduced from the transmembrane proteins to intracellular signaling components. These can be in the form of adaptor proteins lacking any enzymatic activity or they can contain various enzymatic activities such as kinase, phosphatase or lipase activities. Exerting their functions intracellularly, they will propagate and translate the signals into a proper cellular response, a response not only depending on the quality of the signal but also on the quantity and the presence of other incoming signals.
One type of extracellular messengers are the growth factors; polypeptide molecules that are important regulators of many cellular responses, most notably cell proliferation. They exert their effects on the cell by binding to specific receptors on the cell surface, causing dimerization of the receptors. These receptors most commonly contain a tyrosine kinase activity, either intrinsically or they form a complex with an intracellular tyrosine kinase.
Aberrant regulation of growth factors and their receptors has been shown to be involved in many different diseases such as cancer, cardiovascular diseases and developmental defects.
In some cases, the cells produce a growth factor for which they carry the corresponding receptor, resulting in autocrine stimulation of the receptors. In other cases, the receptors are present in too high amounts or carry mutations, rendering them constitutively active even in the absence of growth factor. There are also chromosomal translocations giving rise to fusion proteins between a growth factor receptor and another protein, leading to ligand independent activation of the receptor. It is also possible that a loss of function of growth factor signaling in some cases can lead to disease, e.g. growth factor receptors acting as tumor suppressors.
Learning more about the function and biology of growth factors and their receptors is crucial to the understanding of the mechanisms behind these diseases and for the development of new therapies.
One member of the growth factor family is platelet-derived growth factor (PDGF), which regulates various cellular responses e.g. proliferation and migration and which is implicated in the pathogenesis of several diseases. There are two types of receptors for PDGF, α and β, and upon ligand-induced activation they form different dimeric complexes. This thesis is focused on signal transduction via the receptors for PDGF with the emphasis on the differences in signals transduced by homo- and heterodimeric PDGF receptor complexes.
Platelet-derived growth factor and its receptors
Platelet-derived growth factor
PDGF was originally purified from platelets (Antoniades et al., 1979; Heldin et al., 1979). It is a potent stimulator of growth and migration of mesenchymal cells such as fibroblasts and smooth muscle cells, but is also active on other cell types e.g. neurons and platelets (reviewed in Raines and Ross, 1993).
PDGF is a dimeric molecule consisting of homo- or heterodimers of two structurally related disulfide-linked polypeptide chains, denoted A and B. Three isoforms, PDGF-AA, PDGF-AB and PDGF-BB, exist. In a preparation from human platelets, PDGF-AB was found to be the most abundant isoform making up approximately 70% of the PDGF produced, while most of the remaining 30% consists of PDGF-BB and only very little is PDGF-AA (Hammacher et al., 1988; Hart et al., 1990). Many different cell types maintained in culture in vitro have been shown to produce PDGF, such as macrophages (Shimokado et al., 1985), endothelial cells (DiCorleto and Bowen-Pope, 1983) and fibroblasts (Raines et al., 1989).
Interestingly, the transforming protein v-sis of the simian sarcoma virus is a homologue of the PDGF B-chain (Doolittle et al., 1983; Waterfield et al., 1983), indicating a role for PDGF in oncogenesis.
The mature forms of the PDGF-A- and B-chains are about 100 amino acids long and show about 60% sequence similarity. There are eight cysteine residues that are conserved between the two chains; two of the cysteines (the second and the fourth) are involved in interchain disulfide bonds in the dimers, whereas the other six cysteines form disulfide bonds within the chain (Andersson et al., 1992; Haniu et al., 1994; Haniu et al., 1993). The crystal structure of PDGF-BB has been resolved (Oefner et al., 1992) and shows that the two subunits in the dimer are arranged in an antiparallel manner. Each form a tight knotlike structure from which two loops extend in one direction (loops 1 and 3) and another loop in the opposite direction (loop 2). Because the two subunits are antiparallel, loop 2 in one subunit will be close to loops 1 and 3 of the other subunit. The three-dimensional structure of PDGF- BB resembles that of the related vascular endothelial growth factor (VEGF) (Muller et al., 1997), but also the structures of the more distantly related nerve growth factor (NGF) and transforming growth factor-β (TGF-β) (Murray-Rust et al., 1993). They all contain the characteristic cystine knot structure. Using site-directed mutagenesis, it has been shown that the epitopes important for receptor binding are located in loops 1 and 3, but loop 2 is also involved (Andersson et al., 1995; Clements et al., 1991; Fenstermaker et al., 1993).
The genes for the human PDGF A- and B-chains are localized on chromosome 7 and 22, respectively (Betsholtz et al., 1986; Dalla Favera et al., 1982; Swan et al., 1982). Both chains are synthesized as precursor molecules, which undergo proteolytic cleavage into their mature forms and are secreted as dimers of 30 kDa. The PDGF A-chain exists both as a short and a long form due to differential splicing. The short form is secreted, whereas the long form contains a C-terminal basic sequence that mediates retention inside the cell and to the extracellular matrix (Kelly et al., 1993; LaRochelle et al., 1991; Östman et al., 1991; Raines and Ross, 1992). The B-chain also contains this retention sequence, which causes the PDGF- BB isoform to be mainly cell-associated and only a small fraction secreted.
Recently, a new PDGF isoform, PDGF-C, was identified (Li et al., 2000). Like the A- and B-chains, it forms a disulfide-bonded dimer, PDGF-CC, and contains a PDGF/VEGF- homology domain, located in the C-terminus. In PDGF-C, this domain shows a low overall sequence similarity with other PDGF/VEGF domains, although the eight cysteine residues involved in inter- and intrachain disulfide bonds are conserved. In addition, there are four extra cysteine residues in this domain. In the N-terminus of PDGF-C, there is CUB-domain, which is not observed in the other family members. It was shown that the presence of the CUB-domain inhibited PDGF-CC binding to the PDGF α-receptor and that PDGF-C is
activated by proteolytic removal of this domain. Elucidating the biological functions, PDGF- CC was shown to have a proliferative activity on human foreskin fibroblasts, as well as on cardiac fibroblasts in transgenic mice. A function during kidney development was also suggested.
Receptors for platelet-derived growth factor
To exert its functions, PDGF bind to two structurally related protein tyrosine kinase receptors, denoted α- and β-receptors (Claesson-Welsh et al., 1988; Claesson-Welsh et al., 1989; Matsui et al., 1989a; Yarden et al., 1986). During synthesis, both the α- and the β-receptor are glycosylated to form their mature forms of 170 kDa and 185 kDa, respectively, and are transported to the cell surface. Each receptor consists of an extracellular part with five immunoglobulin-like domains and an intracellular part with tyrosine kinase activity. A single transmembrane domain separates the two parts. For ligand binding, the three outermost Ig- domains are primarily involved (Heidaran et al., 1990; Yu et al., 1994; Lokker et al., 1997).
The intracellular part contains a juxtamembrane domain lacking enzymatic activity, a tyrosine kinase domain that is typically split by an insert of about 100 amino acids and a C-terminal tail. The amino acid sequence similarity between the two receptors is high in the juxtamembrane and kinase domains (~80% identical), and relatively low in the extracellular domain, the kinase insert and the C-terminal tail (~30% identity).
On the basis of sequence similarity and structural characteristics, receptor tyrosine kinases are grouped into subclasses. The presence of a split kinase domain and immunoglobulin-like domains in the extracellular part, places the PDGF receptors in the subgroup III of receptor tyrosine kinases, together with the stem cell factor (SCF) receptor (c- Kit), the colony stimulating factor-1 (CSF-1) receptor and fetal liver kinase-2 (Flk-2) receptor (reviewed in Fantl et al., 1993).
The human α-receptor gene is localized on chromosome 4, close to the genes for c-Kit and vascular endothelial growth factor-2 (VEGF-2) receptor (Spritz et al., 1994) and the human β-receptor gene is located on chromosome 5, close to the CSF-1 receptor gene (Roberts et al., 1988). Thus, the subclass III receptor tyrosine kinase genes are clustered on the chromosomes, indicating a common origin.
Receptors for PDGF were originally identified on fibroblasts, smooth muscle cells and glial cells, but it has later been shown that PDGF receptors are expressed on many different cell types (Raines and Ross, 1993). The availability of PDGF receptors on the target cell is naturally crucial to the PDGF dependent cellular responses and is a subject to regulation.
Treatment of cells with various growth factors such as basic fibroblast growth factor (bFGF) and TGF-β has been shown to affect the expression of PDGF receptors (Heldin and Westermark, 1999). Also pathological processes can affect the expression levels; β-receptor expression on connective tissue cells was found to be increased during inflammation (Rubin et al., 1988a).
Functions of PDGF and PDGF receptors in vivo Cellular effects of PDGF
PDGF was originally identified as a potent mitogen for connective tissue cells, such as fibroblasts, smooth muscle cells and glial cells. Later studies have shown that PDGF is a regulator of many different cellular responses, some of which are discussed below.
Cell proliferation
Treatment of PDGF receptor expressing cells with PDGF for sufficient time periods, leads to cell cycle progression and eventually cell division, with DNA synthesis occurring approximately 8-16 hours after adding PDGF. Both the α- and the β-receptors are capable of inducing a proliferative response (Eriksson et al., 1992), but it is not clear which of the receptors is most mitogenically potent, which is probably due to cell type variations and variations in expression levels of the receptors. In one report, the β-receptor was found to have a higher transforming activity in NIH 3T3 cells cotransfected with ligand and receptor (Heidaran et al., 1993). When α- and β-receptors are expressed together at comparable levels, the αβ-heterodimer has been shown to be the most potent inducer of mitogenicity (Ekman et al., 1999; Emaduddin et al., 1999; Rupp et al., 1994), which may be explained by a difference between the homo- and heterodimers in autophosphorylation pattern and recruitment of downstream signaling molecules (see discussion under ”Signal diversification by homo- and heterodimerization”).
The involvement of different signal transduction molecules and pathways in growth stimulation is an area of intensive research. Both PDGF receptors bind and activate a multitude of downstream signaling components, many of which contain Src Homology 2 (SH2) domains, which bind phosphotyrosine residues, and many of them implicated in the mitogenic response. One signaling pathway of crucial importance for the proliferative response is the Ras/MAP kinase pathway. Overactivity of several components in this pathway has been shown to result in cell transformation (Seger and Krebs, 1995). Activation of Ras by PDGF receptors can occur by direct binding of the adaptor protein complex Grb2/Sos1 to the receptor or by indirect binding of this complex to molecules bound to the receptor, including Shc and SHP-2.
There are also reports suggesting that Ras/MAP kinase-independent pathways are important for PDGF-induced mitogenicity. By microinjection of antibodies against different signaling molecules or SH2 domains acting as dominant inhibitors, it was demonstrated that Nck and phosphatidylinositol 3-kinase (PI3-kinase) are important for DNA-synthesis and can act in a Ras-independent way (Roche et al., 1996; McIlroy et al., 1997). Other signal transduction molecules shown to play a role in PDGF-dependent growth include PLC-γ (Valius and Kazlauskas, 1993) and c-Src (Roche et al., 1995; Twamley-Stein et al., 1993).
It is of vital importance for the cell to regulate and balance the incoming proliferative signals in order to avoid excessive growth. One way of doing this is to recruit the GTPase activating protein of Ras, RasGAP, which will inactivate Ras. The PDGF β-receptor has been shown to use this strategy by recruiting RasGAP to one of its phosphorylated tyrosine residues. Another example of a negative regulator that is recruited to both the α- and β- receptors is the Cbl proto-oncogene, which inhibits PDGF-dependent cell proliferation by enhancing ligand-induced ubiquitination and degradation of the receptors (Miyake et al., 1999). Furthermore, protein tyrosine phosphatases that dephosphorylate the PDGF receptors may negatively regulate the mitogenic response. One reported example is the interaction of the low molecular weight protein tyrosine phosphatase (LMW-PTP) with the PDGF β- receptor, which leads to dephosphorylation of the receptor and decreased mitogenic signaling (Berti et al., 1994). In another report (Klinghoffer and Kazlauskas, 1995), the SH2 domain- containing tyrosine phosphatase SHP-2 was found to in vitro dephosphorylate certain phospotyrosine residues in the PDGF β-receptor, which potentially could have a negative influence on the mitogenic response.
Another factor influencing the proliferative response to PDGF and other growth factors is the engagement of integrins. Activated PDGF β-receptor was found to associate with the αvβ3 integrin, and plating cultured cells on vitronectin, a ligand for the αvβ3 integrin,
potentiated the mitogenic response to PDGF-BB. This correlated with an increased activation of MAP kinase (Schneller et al., 1997).
Cell motility
PDGF stimulation causes rearrangements of the actin cytoskeleton and induces cell movement. These responses includes the loss of stress fibres and the appearance of edge ruffles around the cell margins. Similar structures are seen on the leading edge of migrating cells. In some cell types, it is also possible to see the formation of circular ruffles, which are large, circular actin structures (Mellström et al., 1988). The role of these circular ruffles in cell movement is not clear. Treatment with PDGF induces two types of cell movements;
chemokinesis or random migration, and chemotaxis or directed migration. Chemotaxis is of physiological importance, e.g. in wound healing and embryonic development, whereas the physiological role of chemokinesis remains to be clarified.
The two different PDGF receptors have different chemotactic properties. Cells expressing the PDGF β-receptor migrate towards PDGF-BB (Kundra et al., 1994), whereas the PDGF α-receptor mediates chemotaxis only in certain cell types (Ferns et al., 1990;
Hosang et al., 1989; Matsui et al., 1989b). In monocytes and fibroblasts (Siegbahn et al., 1990), which express endogenous α-receptors, or in porcine aortic endothelial (PAE) cells transfected with α-receptors (Eriksson et al., 1992), PDGF-AA (which specifically activates PDGF α-receptors) failed to induce a chemotactic response. If co-expressed with the β- receptor, the PDGF α-receptor even inhibits the β-receptor-mediated chemotactic response in some cell types, such as vascular smooth muscle cells (Koyama et al., 1992), fibroblasts and monocytes (Siegbahn et al., 1990). One explanation for the contradictory results on the chemotactic properties of the PDGF α-receptor, may be the different methodological designs used to measure the chemotactic response, where some methods may measure chemokinesis to a higher degree than others. Still, it has been demonstrated that the PDGF α-receptor transduces inhibitory signals for chemotaxis, overriding the intrinsic capability of the α- receptor to stimulate migration. In contrast to the wildtype receptors, mutant PDGF α- receptors, in which either of the tyrosine residues 768, 993 or 1018 had been replaced by phenylalanine residues, were able to induce a chemotactic response in response to PDGF (Yokote et al., 1996b).
A key molecule in mediating the chemotactic response is PI3-kinase. Mutant PDGF β- receptors, in which the PI3-K binding sites Tyr740 and Tyr751 have been removed by changing them to phenylalanine residues, lose their ability to induce chemotaxis and edge ruffling in response to PDGF (Kundra et al., 1994; Wennström et al., 1994b). The same effect is achieved by using a dominant negative form of the regulatory subunit p85 or the PI3-K inhibitor wortmannin (Hooshmand-Rad et al., 1997; Wennström et al., 1994a). Most likely, PI3-K exerts its effect on migration by activating members of the Rho family of small GTP binding proteins. One of the members, Rac, has been to shown to be activated via the PI3-K signaling pathway (Hawkins et al., 1995) and plays a role in the formation of edge ruffles (Ridley et al., 1992) and chemotaxis (Hooshmand-Rad et al., 1997). Other downstream effectors of PI3-K shown to be involved in the motility response include members of the PKC family of serine/threonine kinases. They can be directly activated by the PI3-K lipid product PI(3,4,5)P3 (Derman et al., 1997).
PI3-K-independent signaling pathways have also been reported to be important for the chemotactic response. A mutant PDGF β-receptor, in which the binding site for phospholipase C-γ1 (PLC-γ1) had been removed, showed a decreased chemotactic response to PDGF, when expressed in CHO cells (Kundra et al., 1994). However, expressing the same mutant receptor in PAE cells, did not have any effect on PDGF stimulated chemotaxis
(Wennström et al., 1994b). In another study, a Src phosphorylation site in the PDGF β- receptor, Tyr 934, was mutated to a phenylalanine residue. This mutant receptor showed an increased phosphorylation and activation of PLC-γ1, which correlated with a higher PDGF- induced chemotactic response (Hansen et al., 1996). Using overexpression of wildtype PLC- γ1, it was further strengthened that PLC-γ1 plays a positive role in the chemotactic response to PDGF (Rönnstrand et al., 1999b).
An involvement of Ras in chemotaxis has also been implicated. Using cells expressing a dominant negative Ras, it was found that Ras inhibition suppresses migration toward PDGF- BB, but does not affect fibronectin-induced chemotaxis. Cells producing a constitutively active form of Ras also showed impaired chemotaxis, showing that either too little or too much Ras activity negatively affects the chemotactic response (Kundra et al., 1995).
Furthermore, abrogating the binding of the negative regulator of Ras, RasGAP, to the PDGF β-receptor leads to an increased ligand-induced migratory response (Kundra et al., 1994). The downstream effectors of Ras mediating migration have been studied and imply an involvement of MAP kinase-independent pathways, since expression of a dominant negative form of MEK-1 did not have any affect on PDGF-stimulated chemotaxis (Anand-Apte et al., 1997). It has been demonstrated that activated Ras interacts with the catalytic domain of PI3- K (Rodriguez-Viciana et al., 1994), thus linking Ras to signaling pathways involved in the regulation of the actin cytoskeleton. Indeed, a direct role of PI3-K in Ras regulated control of actin organization was found (Rodriguez-Viciana et al., 1997).
It is possible that RasGAP affects cell movement independent of its effect on Ras, since RasGAP forms a complex with p190RhoGAP, which through its ability to inactivate RhoGTPases may control the actin cytoskeleton and migration. RasGAP also interacts with the 62 kDa adaptor protein Dok, a protein reported to enhance the cell migratory response to insulin (Noguchi et al., 1999).
The Src family of protein tyrosine kinases is a group of cytoplasmic tyrosine kinases that phosphorylates and regulates a plethora of proteins, many of them implicated directly or indirectly in the regulation of the actin cytoskeleton (reviewed in in Abram and Courtneidge, 2000), which makes Src family kinases likely regulators of PDGF-induced cell movement.
However, a Src binding mutant of the PDGF α-receptor expressed in PAE cells, still had the ability to induce actin reorganization and chemotaxis (Hooshmand-Rad et al., 1998).
Other regulators of the actin cytoskeleton include cytoplasmic protein tyrosine phosphatases. One of them, SHP-2, is a binding partner for the PDGF receptors. Abrogating the binding of SHP-2 to the PDGF β-receptor leads to an impaired chemotactic response, possibly involving a decreased activity of focal adhesion kinase (FAK) (Qi et al., 1999;
Rönnstrand et al., 1999a). The low molecular weight protein tyrosine phosphatase (LMW- PTP), when overexpressed in NIH 3T3 cells, potentiates the chemotactic response to PDGF via dephosphorylation of its target proteins, including p190RhoGAP (Chiarugi et al., 2000).
Other effects
PDGF is an inducer of differentiation in PC12 neuronal cells (Heasley and Johnson, 1992), a response that persists in cells expressing receptor mutants defective for mitogenesis (Vetter and Bishop, 1995). FDC-12 myeloid progenitor cells containing PDGF β-receptors undergo monocytic differentiation in response to PDGF requiring the activity of PLC-γ1 (Alimandi et al., 1997). In another report, different PDGF β-receptor mutants were investigated for their ability to induce voltage-dependent sodium (Na+) channel expression, which is a marker for neuronal differentiation in PC12 cells. Src family kinases were implicated in this response, since removal of the two juxtamembrane tyrosine phosphorylation sites responsible for binding of Src, inhibited the induction of Na+ channels (Fanger et al., 1997).
PDGF not only stimulates cells to divide, but also prevents cells from undergoing programmed cell death, apoptosis. PC12 cells expressing PDGF β-receptors were protected from apoptosis by the addition of PDGF and this effect involved the activation of PI3-K (Yao and Cooper, 1995). Moreover, PDGF has also been shown to protect oligodendrocytes from apoptosis (Barres et al., 1992). In contrast, serum-deprived normal rat kidney fibroblasts undergo apoptosis when stimulated with PDGF (Kim et al., 1995).
Extracellular matrix components have been shown to affect the behavior of cells via activation of integrins. PDGF is known to stimulate the production of various extracellular matrix molecules, such as fibronectin, hyaluronic acid, collagen and collagenase (Bauer et al., 1985; Blatti et al., 1988; Canalis et al., 1981; Heldin et al., 1989). Consequently, PDGF may modulate its biological functions indirectly via deposition of extracellular matrix and activation of integrins, a hypothesis that is further strenghtened by the finding that PDGF also induces the synthesis of the collagen binding integrin α2 chain in fibroblasts (Åhlén and Rubin, 1994).
Physiological role of PDGF and PDGF receptors
Embryonic development
PDGF and its receptors are expressed in the developing embryo (Mercola et al., 1990;
Morrison-Graham et al., 1992; Orr-Urtreger and Lonai; 1992; Schatteman et al., 1992) and in the placenta (Goustin et al., 1985), suggesting a regulatory role in embryogenesis. PDGF-A and PDGF α-receptor are present in the early stages of embryogenesis, whereas PDGF-B and PDGF β-receptor are produced at later stages. PDGF receptors are often expressed in mesenchymal structures and their ligands often found in adjacent epithelium, pointing to a paracrine mode of action (Ataliotis and Mercola, 1997).
The inactivation of the genes for the PDGF-A chain (Boström et al., 1996) and the PDGF-B chain (Levéen et al., 1994) as well as for the PDGF α-receptor (Soriano, 1997) and the PDGF β-receptor (Soriano, 1994) all lead to embryonic or perinatal death and has established the importance of PDGF signaling in embryonic development. Gene targeting of the B-chain or the β-receptor gives very similar phenotypes. In both cases the mice die perinatally. Kidney development is severely affected with a total absence of mesangial cells in the glomeruli, leading to defective filtration. Hematological disturbances include anemia and thrombocytopenia. Cardiovascular defects are observed with dilatation of the heart and large arteries, as well as the formation of capillary microaneurysms, leading to severe hemorrhages. This may be explained by the failure of blood vessels to attract pericytes, which normally contribute to the mechanical stability of the vessel wall (Lindahl et al., 1997a). In addition, the B-chain knock-out mice show heart defects in the form of hypertrophy and trabeculation of the myocardium, suggesting that the B-chain may act through the α-receptor during heart development.
Absence of the PDGF A-chain causes approximately 50% of the mice to die before embryonic day 10 (E10), whereas the remaining mice survive for a longer time, but all die before 6 weeks of postnatal age. The phenotype is characterized by impairment of lung development, resulting in severe emphysema. This picture can be explained by a defective alveogenesis due to lack of spreading of lung smooth muscle cell progenitors to the alveoli (Lindahl et al., 1997b).
Disruption of the PDGF α-receptor gene shows an earlier and more severe phenotype than the A-chain knock-out, leading to embryonic death. Neural crest development is impaired, resulting in incomplete cephalic closure and there is a deficiency in myotome formation, causing abnormal patterning of the somites. The naturally occuring mouse mutant Patch involves a deletion of the α-receptor gene (Stephenson et al., 1991) and exhibits a
similar phenotype, although additional defects are observed probably due to the involvement of the neighboring gene for the SCF receptor. The discrepancy between the A-chain and the α-receptor knock-outs may be due to partial compensation by other ligands; in the A-chain knock-out, the B-chain or the C-chain can still activate the α-receptor.
It is interesting to note that PDGF A- and B-chains both seem to be important for the development of specific subsets of smooth muscle cells. PDGF B-chain-deficient embryos lack pericytes and mesangial cells, and the PDGF A-chain knock-outs lack alveolar myofibroblasts, suggesting a role in the development of (Lindahl and Betsholtz, 1998).
Wound healing
Wound healing is a complex, but well-orchestrated process including cell migration, proliferation and extracellular matrix production and involving many different cell types.
PDGF is one of the growth factors implicated in the regulation of these processes.
At early stages of a wound, PDGF is released at the site of injury by platelets in the blood clot. At later stages, PDGF is also secreted from other cell types, such as activated macrophages, thrombin-stimulated endothelial cells, smooth muscle cells of damaged arteries, fibroblasts and epidermal keratinocytes. PDGF acts as chemoattractant for a variety of cells, including neutrophils and macrophages in the earlier stages of wound healing, as well as fibroblasts and smooth muscle cells in the formation of granulation tissue. It also stimulates the proliferation of fibroblasts and smooth muscle cells. The expression of PDGF receptors is low in resting tissue, but PDGF β-receptor expression is markedly upregulated in inflammatory tissue, increasing its responsiveness to ligand (Reuterdahl et al., 1993; Rubin et al., 1988b). Furthermore, PDGF activates macrophages to release other growth factors of importance for the healing process, e.g. fibroblast growth factor (FGF), and stimulates fibroblasts to produce extracellular matrix molecules, such as fibronectin and hyaluronic acid, which is important in the formation of the granulation tissue. PDGF may also play a role in wound contraction, since it stimulates contraction of collagen matrices in vitro (Clark et al., 1989; Gullberg et al., 1990), a response shown to be dependent on the activation of PI3-K (Heuchel et al., 2000), and in remodeling of the wound through the production of collagenase (reviewed in Heldin and Westermark, 1996).
Another important part of wound healing is neovascularization. PDGF has been shown to have a weak angiogenic activity (Risau et al., 1992), and in one report, PDGF-BB stimulated the generation of functional new blood vessels in a rat skin flap model (Brown et al., 1995). Receptors for PDGF are absent from endothelial cells in larger vessels, but have been demonstrated on capillary endothelial cells (Bar et al., 1989; Smits et al., 1989) and on microvascular pericytes (Sundberg et al., 1993), suggesting a direct role for PDGF in vascularization. It is also possible that PDGF stimulate angiogenesis in an indirect way by inducing the secretion of other angiogenic growth factors (Sato et al., 1993). The disruption of the genes for PDGF-B and PDGF β-receptor shows that vascular endothelium can still be formed in the absence of PDGF-B/PDGFRβ signaling, but that the integrity of the vessel walls is defect, resulting in severe hemorrhages.
In vivo studies have demonstrated that application of PDGF enhances the formation of granulation tissue (Grotendorst et al., 1985; Sprugel et al., 1987) and that PDGF-BB is significantly more effective than PDGF-AA in this respect (Lepistö et al., 1992). Clinical studies have shown positive effects on the healing of decubitus ulcers (Robson et al., 1992), as well as improved healing in patients with decreased healing capacity, such as diabetics (Steed and the Diabetic Ulcer Study Group, 1995).
PDGF in diseases
Malignancies
The first indications that PDGF is involved in tumorigenesis came with the discovery that the sis oncogene product was homologous to the PDGF B-chain (Doolittle et al., 1983;
Waterfield et al., 1983) and that sis-transformation occurs via stimulation of PDGF receptors in an autocrine manner (reviewed by Westermark et al., 1987). This stimulated further studies on the role of PDGF in malignancies and has revealed that PDGF and PDGF receptors are expressed in several human tumors (reviewed in Heldin and Westermark, 1999).
In human glioblastomas, the expression pattern provides evidence for two autocrine loops: PDGF A-chains and PDGF α-receptors are present in tumor cells, whereas PDGF B- chains and PDGF β-receptors are expressed in the stromal compartment (Hermanson et al., 1988; Plate et al., 1992). Interestingly, overexpression of PDGF and its receptors was particularly observed in the more malignant, high grade tumors (Westermark et al., 1995).
The importance of the PDGF signaling system for glioma cell growth was underscored by the findings that dominant negative mutants of PDGF (Shamah et al., 1993) or a truncated PDGF β-receptor, acting in a dominant negative manner (Strawn et al., 1994), could inhibit the growth of glioma cells. Another form of intracranial tumors with a possible autocrine mode of PDGF stimulation are meningiomas, which have been shown to co-express PDGF B-chains and PDGF β-receptors and that contain activated β-receptors, as demonstrated by the use of antibodies directed against phosphorylated Tyr751 in the β-receptor (Shamah et al., 1997).
Consistent with PDGF being a connective tissue mitogen, PDGF and its receptors were found to be expressed in a malignancy-dependent manner in fibromas and fibrosarcomas (Alman et al., 1992; Smits et al., 1992).
Tumors of epithelial cell origin have also been shown to express PDGF and/or its receptors, although normal cells in these tissues do not respond to PDGF. In some cases, a possible autocrine loop may occur by the concomitant expression of PDGF and its receptors, e.g. in thyroid carcinoma (Heldin et al., 1991; Heldin et al., 1988), carcinomas of the lung (Antoniades et al., 1992), breast carcinoma (Coltrera et al., 1995) and gastric carcinoma (Chung and Antoniades, 1992). In other cases, an effect of PDGF production on the stromal compartment is likely (Chaudhry et al., 1992; Forsberg et al., 1993; Lindmark et al., 1993).
A direct role of PDGF and PDGF receptors in oncogenesis has come with the findings of structural aberrations of the corresponding genes, leading to overexpression or expression of an abnormal protein. Chronic myelomonocytic leukemia is associated with a translocation between chromosomes 5 and 12, resulting in a fusion protein between the Ets-like transcription factor Tel and the kinase domain of the β-receptor (Golub et al., 1994). It is likely that Tel-mediated oligomerization leads to constitutive activation of the β-receptor, causing cell transformation. This is supported by data showing that overexpression of the Tel part of Tel-PDGFRβ causes a reduction of tyrosine phosphorylation of the fusion protein (Sjöblom et al., 1999). Another cytogenetic abnormality in chronic myelomonocytic leukemia that results in a transforming protein is the fusion between the Huntingtin interacting protein 1 (HIP1) and the transmembrane and tyrosine kinase domains of PDGF β-receptor (Ross et al., 1998). In dermatofibrosarcoma protuberans and giant-cell sarcoma, the PDGF B-chain gene is fused to the collagen gene COL1A1, leading to transforming activity (Simon et al., 1997).
Transformation by the bovine papilloma virus has been suggested to involve activation of the PDGF β-receptor. This virus causes fibropapillomas, benign skin tumors, mediated by the transforming protein E5, which has been shown to interact with the transmembrane and juxtamembrane domains of the PDGF β-receptor forming stable complexes. This leads to ligand-independent dimerization and activation the receptors (Nilson and DiMaio, 1993; Petti and DiMaio, 1994).
Atherosclerosis
The atherosclerotic process is initiated by mechanical or non-mechanical injury to the endothelium, leading to recruitment of activated monocytes into the subendothelial space (intima). The monocytes change into macrophages, accumulate lipids and become foam cells, forming fatty streaks. Moreover, vascular smooth muscle cells migrate from the medial layer into the intimal layer, where they proliferate, and eventually an atherosclerotic plaque is formed. The expression of PDGF by smooth muscle cells and and macrophages has been shown to be increased in atherosclerotic plaques (Barrett and Benditt, 1988; Ross et al., 1990;
Wilcox et al., 1988), and the expression of PDGF β-receptor in intimal smooth muscle cells is increased, leading to enhanced responsiveness to PDGF-BB (Rubin et al., 1988b). The PDGF secreted by activated macrophages, smooth muscle cells, endothelial cells or released from platelets in thrombi acts by chemotactically attracting the smooth muscle cells into the intima and consequently, PDGF signaling is responsible for the increased pool of smooth muscle cells, which is an important feature of intimal thickening.
The role of PDGF in the atherosclerotic process is further supported by in vivo studies of restenosis models, in which the artery is injured by balloon angioplasty (similar technique is used in percutaneous transluminal corronary angioplasty (PTCA) in patients). Also in this case, vascular smooth muscle cells are recruited to the intima leading to intimal thickening and restenosis. Using a rat carotid artery model, an increased amount of PDGF receptors was observed in the vessel wall (Abe et al., 1997; Panek et al., 1997), but the intimal thickening could be inhibited by a neutralizing antibody to PDGF (Ferns et al., 1991). A PDGF-specific tyrosine kinase inhibitors has also been shown to halt the neointima formation in a porcine restenosis model (Banai et al., 1998). In contrast, infusion of PDGF-BB into rats after carotid angioplasty promoted smooth muscle migration and intimal thickening (Jawien et al., 1992).
The two types of PDGF receptors seem to play different roles in the vascular wall. In contrast to the β-receptor, the activated α-receptor inhibits the migration of vascular smooth muscle cells and could thus be a regulator of intimal thickening (Koyama et al., 1992).
Furthermore, PDGF-BB stimulates DNA synthesis in vascular smooth muscle cells, whereas PDGF-AA does not (Inui et al., 1994), strenghtening the role of PDGF-AA as a modulator of cell activity in the arterial wall.
Fibrosis
Consistent with PDGF being an important stimulator of connective tissue formation, overactivity of PDGF has been reported in several diseases involving fibrosis. Myelofibrosis, e.g. bone marrow fibrosis, is associated with increased levels of PDGF in plasma (Gersuk et al., 1989). In liver fibrosis and scleroderma (systemic sclerosis), which involves fibrosis of the skin and various visceral organs, induction of PDGF β-receptors has been demonstrated (Klareskog et al., 1990; Wong et al., 1994). In idiopathic or injury-induced pulmonary fibrosis, the PDGF α-receptor seems to play an important pathophysiological role. Lung fibroblasts normally express PDGF β-receptors and only low levels of the α-receptor, but during pulmonary fibrosis PDGF α-receptors are markedly upregulated, leading to increased responsiveness to PDGF-AA (Bonner et al., 1993). Interestingly, the induction of α-receptors in the lung fibroblasts is required for maximal chemotactic and mitogenic responses to all isoforms of PDGF (Coin et al., 1996; Osornio-Vargas et al., 1996), indicating that unique signaling via the αβ heterodimeric receptor complex could potentiate these responses. The source of PDGF during pulmonary fibrosis are the alveolar macrophages, which have been shown to produce increased amounts of PDGF, but also the alveolar epithelium which starts to produce PDGF during fibrosis.
Inflammation
Chronically inflamed synovia, ocurring in joints of patients with rheumatoid arthritis, has a clear upregulation of PDGF β-receptors, both in vascular and stromal cells (Reuterdahl et al., 1991; Rubin et al., 1988a). Furthermore, PDGF is present in high amounts in inflamed joints (Sano et al., 1993) and thus, PDGF signaling could play a role in the pathogenesis of chronic arthritis by driving proliferation of synovial cells.
Glomerulonephritis is another inflammatory disorder in which PDGF is implicated. This disease is characterized by massive mesangial cell proliferation and extracellular matrix production, leading to destruction of the filtrating glomeruli. Mesangial cells express both PDGF and its receptors, and PDGF signaling is thought to be responsible for the disordered expansion of the mesangial cells, whereas the extracellular matrix deposition is primarily affected by other factors, such as TGF-β. In line with this, kidneys in humans with mesangial proliferative nephritis have an increased expression of PDGF and induction of PDGF-B has been shown to induce glomerulonephritis by stimulating mesangial cell proliferation.
Moreover, in an experimental model for glomerulonephritis, expression of PDGF and PDGF β-receptor correlated with the mesangial cell proliferation. Administration of neutralizing PDGF antibodies in this model decreased the proliferation of mesangial cells. Apart from the mesangial cells themselves, macrophages in the inflamed kidney could be a source of PDGF and contribute to the expansion of mesangial cells. Thus, both autocrine and paracrine mechanisms may be involved (reviewed in Heldin and Westermark, 1996).
Signal transduction via PDGF receptors
Receptor activation
Dimerization has been shown to be a general mechanism for the activation of cell surface receptors, including PDGF receptors (reviewed in Heldin, 1995). The dimeric nature of PDGF allows it to interact simultaneously with two receptors, inducing receptor dimerization. The PDGF α-receptor binds both the A-chain and the B-chain with high affinity, whereas the PDGF β-receptor only binds the B-chain. Consequently, PDGF-AA induces αα-receptor homodimers, PDGF-AB recruits αα-receptor homodimers and αβ-receptor heterodimers, whereas PDGF-BB can form all possible receptor dimers (αα-, αβ- and ββ-receptor dimers).
The epitopes in the receptor most important for ligand binding resides in the three outermost immunoglobulin-like domains, but immunoglobulin-like domain 4-mediated receptor-receptor interactions have also been shown to contribute to dimerization (Omura et al., 1997). A similar mechanism for the fourth immunoglobulin-like domain has also been found in dimerization of SCF receptors (Blechman et al., 1995).
Dimerization brings the two receptor kinases in the dimer close to each other, allowing autophosphorylation of tyrosine residues in the receptors. This phosphorylation is thought to occur mainly in trans (Emaduddin et al., 1999; Kelly et al., 1991; Vaillancourt et al., 1996;
see paper II in “Present Investigations”). There are speculations whether dimerization is sufficient to activate the receptor or if the proper ligand is needed to induce additional conformational changes. Several tyrosine kinase receptors can be dimerized and activated by binding of receptor specific antibodies, e.g. PDGF receptors (Rönnstrand et al., 1988), whereas Fab fragments are inactive. Introduction of an inter-receptor disulfide bond in the EGF receptors resulted in constitutively active dimeric receptors (Sorokin et al., 1994).
Furthermore, the Neu (ErbB2) oncogene product contains a point mutation (V664E) in the juxtamembrane domain leading to constitutive dimerization and cell transformation. These data argue that dimerization is enough for activation of receptors. On the other hand, other
αααα αααα αααα ββββ ββββ ββββ
B A A
A B B
A B
B B
B B
Fig. 1 Ligand-binding specificities of the PDGF receptors.
The abilities of the different PDGF isoforms to induce the formation of homo- and heterodimeric receptor complexes is depicted.
C C
18
findings support a need for ligand-induced conformational changes, exemplified by the Epo receptor and the ErbB2/Neu receptor. In these cases, unliganded dimers may exist, but become fully active only upon ligand binding (reviewed in Jiang and Hunter, 1999).
Functionally, the autophosphorylation sites can be divided into two different groups. The first group consists of a tyrosine residue in the activation loop of the kinase domain, the phosphorylation of which leads to an upregulation of the kinase activity. In the PDGF β- receptor, this site corresponds to Tyr857 and mutation of this tyrosine residue results in a dramatic decrease in the kinase activity of the receptor (Kazlauskas et al., 1991). This tyrosine residue is well-conserved in almost all tyrosine kinases and is represented by Tyr849 in the PDGF α-receptor. Analyses of the crystallographic structure of the insulin receptor kinase domain has revealed an important autoinhibitory role for this tyrosine residue (Tyr1162); in the unphosphorylated state, Tyr1162 is bound in the active site competing with protein substrates for the binding and upon autophosphorylation of Tyr1162 (and Tyr1158/1163), there are dramatic conformational changes in the activation loop permitting access to the active site for protein substrates and ATP. In contrast to the case of PDGF β-receptor, substitution of Tyr1162 with phenylalanine in the insulin receptor results in an increased basal kinase activity, consistent with an autoinhibitory role for Tyr1162. In the FGF receptor 1, crystallographic studies reveal a different mechanism of autoinhibition. Neither of the activation loop tyrosines, Tyr653 and Tyr654, is bound in the active site and instead, non- tyrosine interactions are involved in interfering with protein substrate binding, but not with ATP binding. It is to be noted that not all tyrosine kinases are regulated by phosphorylation in the kinase domain; the conserved tyrosine in the kinase domain of the EGF receptor does not appear to be autophosphorylated. How is then trans-autophosphorylation of autoinhibited tyrosine kinase domains initiated? Data from the crystallization of both the IRK and FGFR1K indicate that segments of the activation loop are relatively mobile and this would allow for the existence of an equilibrium between different conformations of the activaton loop in vivo, a majority of these being inhibited. Upon ligand binding, the local concentration of receptors increases, raising the probability of trans-phosphorylation events and shifting the equilibrium.
It is likely that the equilibrium is balanced to provide inhibition strong enough to avoid phosphorylation of substrates in the absence of ligand, but weak enough to permit trans- autophosphorylation of receptors in a dimer (reviewed in Hubbard et al., 1998). Interestingly, many receptor tyrosine kinases can be activated in the absence of ligand by tyrosine phospatase inhibitors such as vanadate, indicating that autoinhibition is not enough to keep the receptors silent.
The other group of tyrosines are usually located outside the kinase domain and in their phosphorylated forms, they provide binding sites for downstream signal tranduction molecules containing Src-homology 2 (SH2) domains or in some cases phosphotyrosine binding (PTB) domains (Pawson, 1995; see further discussion in ”Protein modules in signal transduction molecules”). Since the amino acids surrounding the phosphotyrosine are important in the recognition of SH2 domains, specificity in the binding can be achieved. The PDGF α- and β-receptors contain at least 8 and 11 autophosphorylation sites, respectively, that take part in the binding of many different signal transduction molecules. Some of these molecules are activated by mere binding to the receptor that induces coformational changes, others are phosphorylated by the receptor kinase and thereby activated. There are data indicating that also activation loop tyrosines can bind SH2 domain-containing molecules; the adaptor protein Grb2 binds to the activation loop tyrosines in the TrkA nerve growth factor receptor and the Jak binding protein JAB interacts with the activation loop tyrosine of the janus kinase 2 (Jak2) (MacDonald et al., 2000; Yasukawa et al., 1999). However, so far there are no data showing binding of SH2 domain proteins to the activation loops of the PDGF receptors. The recruitment and clustering of molecules to the cell membrane, provided by the activated receptor, in many cases enables the signal transduction molecules to interact with
the next component in the signaling pathway, being another protein or components of the cell membrane.
There are certain tyrosines that may be phosphorylated by a kinase other than the receptor kinase. One example is the phosphorylation of Tyr934 in the kinase domain of the PDGF β-receptor, which has been shown to be mediated by the Src kinase. In this case, the Src kinase recruited to the receptor modulates the behavior of the receptor itself; replacement of Tyr934 with a phenylalanine residue leads to increased chemotaxis, but decreased mitogenicity in response to PDGF (Hansen et al., 1996). It is possible that Tyr934 is a better substrate for Src than for the receptor kinase, since cytoplasmic tyrosine kinases in general seem to phosphorylate different sequences compared to receptor tyrosine kinases, with the major difference in sequence being at positions P-1 and P+1 (Songyang et al., 1995b). In support of this model is the finding that a naturally occurring mutation (M918T) in the Ret receptor tyrosine kinase, causing the dominantly inherited cancer multiple endocrine neoplasia 2B (MEN2B), results in a switch in the substrate specificity to that of cytoplasmic tyrosine kinases (Songyang et al., 1995a).
Protein modules in signal transduction molecules
In the propagation of signals from the cell surface and into the cell, protein-protein and protein-lipid interactions have been shown to be crucial. The mediators of these interactions are conserved blocks of polypeptides or protein domains. These protein domains include the Src-homology 2 (SH2), Src-homology 3 (SH3), phosphotyrosine binding (PTB), pleckstrin homology (PH) and PDZ domains, and these will be discussed in further detail below. Protein domains or modules are found in most signal transduction molecules and it is estimated that there are hundreds of families of independent protein domains, implying their importance in signaling. In many cases, cytoplasmic signaling proteins contain several different domains, which can have various functional consequencies. Two domains may interact with two different sites on the same molecule increasing the strength of interaction, as is the case of tandem SH2 domains (Ottinger et al., 1998). Separate domains can also bind to distinct partners, allowing complexes of several proteins to be formed. An example of this are adaptor proteins containing SH2 and SH3 domains, e.g. Grb2, which can connect several proteins to each other. Another function is the intramolecular regulation of enzyme activity found in some proteins. The interactions between domains within the same protein then represses enzyme catalysis, as occurs in the Src family kinases and the SHP-2 tyrosine phosphatase.
The Src homology 2 (SH2) domain is a conserved region of about 100 amino acids that was originally identified on the basis of homologies between Src family kinases and several other signaling molecules and that was different from the enzymatic part of Src. Many signaling proteins contain one or two copies of SH2 domains and it is the largest family of domains that can recognize phosphotyrosine residues. Sequence comparison and mutational analyses have identified the highly conserved FLVR motif in SH2 domains as important for phosphotyrosine binding, with the arginine (R) being the only invariant residue (Pawson, 1995). This arginine is located at the base of the binding pocket for the phosphotyrosine and explains why SH2 domains bind phosphotyrosine but not phosphoserine/phosphothreonine.
Only the phosphotyrosine is long enough to reach the arginine and interact with it. It has also been demonstrated that phosphatidylinositol lipid products of PI3-K can bind to SH2 domains of various proteins, e.g. that of PLC-γ1 (Rameh et al., 1998). Structural studies of SH2 domains from several different proteins have revealed a conserved folding, consisting of a central β-sheet flanked by two α-helices. Peptide substrates bind with the phosphotyrosine residue in a conserved pocket on one side of the sheet and with residues C-terminal to the phosphotyrosine in a pocket or groove on the opposite side of the central sheet. This explains the fact that the specificity in SH2 domain interactions is determined by the 3-6 amino acids C-terminal to the phosphotyrosine residue (Songyang et al., 1993).
Based on structural data and known consensus motifs, the SH2 domains fall into two major groups. Group I, Src-like SH2 domains select for charged residues at positions +1 and +2 and a hydrophobic residue at the +3 position, fitting the side chain of this into a hydrophobic pocket. Group III, PLC-γ-like SH2 domains recognize at least five, mainly hydrophobic residues carboxy-terminal to the phosphotyrosine, which fit into a hydrophobic groove of the peptide binding surface. The Grb2 SH2 domain is unique and does not fall into any of these categories; it favors an asparagine at the +2 position. Interestingly, it is possible to change the specificty of SH2 domains by single point mutations, e.g. converting a group III SH2 domain to a group I SH2 domain(Songyang et al., 1995b). This flexibility may have evolutionary advantages, allowing specificity of SH2 domains to change rapidly and the emergence of novel interactions.
Functionally, the SH2 domain-containing molecules can be divided into three categories;
those with an intrinsic enzyme activity, such as PLC-γ, SHP-2, Src and RasGAP, those lacking enzyme activity, instead serving as adaptor proteins, such as Grb2, Crk, Shc, Nck, and the p85 regulatory subunit of PI3-K, and those acting as transcription factors, i.e. Stat proteins. All the above mentioned signaling molecules have been shown to interact with and be activated by the PDGF α- and β-receptors. Two of them show preferential binding to one of the receptors; Crk binds only to the α-receptor and RasGAP only to the β-receptor, which contributes to the different signaling properties of the two receptors.
Another domain specifically recognizing phosphotyrosine-containing motifs is the phosphotyrosine binding (PTB) domain. Unlike SH2 domains, the PTB domain requires residues amino-terminal to the phosphotyrosine for its binding (Zhou et al., 1995), prefering NPXY motifs. Examples of signaling molecules containing a PTB domain are Shc, IRS-1 and Shb. Some PTB domains can also bind unphosphorylated sequences; the adaptor protein FRS2 binds via its PTB domain to a phosphorylated sequence in the TrkA neurotrophin receptor, but to a totally different non-phosphorylated sequence in the FGF receptor.
Strikingly, structural studies show that PTB domains have a similar folding as PH domains, which instead bind phospholipids. To date, no PTB domain is known to bind to any of the PDGF receptors. The Cbl protein, which binds both to the PDGF α- and β-receptors (Bonita et al., 1997; Miyake et al., 1999), has been reported to contain a PTB domain, but recent data suggest the existence of a distantly related but genuine SH2 domain in Cbl (Meng et al., 1999), making it possible that this SH2 domain mediates the binding to the PDGF receptors.
It is likely that there are other families of phosphotyrosine recognition domains remaining to be identified. For example, the adaptor protein Gab-1 contains a C-terminal proline-rich domain capable of binding to the Met receptor in a tyrosine phosphorylation- dependent manner (Weidner et al., 1996).
The pleckstrin homology (PH) domain is a structurally conserved motif of about 120 amino acids with low sequence homology between its members. It was originally identified in the pleckstrin protein, the major protein kinase C substrate in platelets, but has now been found in various kinds of proteins, such as protein kinases (Akt/PKB, Btk and the β- adrenergic receptor kinase, βARK), guanine nucleotide exchange factors for small GTPases (Sos and Dbl), PLC isoforms (β, γ and δ) and GTPase activating proteins (e.g. RasGAP). To exert their functions, PH domain-containing proteins generally require membrane association and indeed, the major binding partner for PH domains are phospholipids, which thus could provide membrane targeting. For instance, the PLCδ1 PH domain binds PI(4,5)P2 and the PH domain in Akt/PKB binds PI(3,4)P2 (Garcia et al., 1995; Franke et al., 1997). This allows for inducible membrane targeting through the production of lipid second messengers. The lipid products of PI3-K play a central role here, and it has been shown that the PH domains of the PDK1 and Akt/PKB serine/threonine kinases bind PI(3,4,5)P3, promoting their membrane association and activation. In another report, PLC-γ was shown to be activated in a PI3-K- dependent manner through PH domain-mediated membrane targeting (Falasca et al., 1998).
PH domains have also been reported to interact with proteins, including the βγ subunits of heterotrimeric G proteins and protein kinase C (PKC). The importance of PH domains in signaling is further strengthened by the finding that mutations in the PH domain of the cytoplasmic tyrosine kinase Btk cause the immunodeficiency disease X-linked agammaglobulinemia in humans (Vihinen et al., 1995).
The Src homology 3 (SH3) domains consist of approximately 60 amino acids and are found in many cytoplasmic signal transduction molecules, but also in other proteins such as cytoskeletal components. They are major mediators in clustering signaling complexes and targeting them to defined cellular compartments. SH3 domains recognize proline-rich sequences with the consensus motif PXXP, that form a type II left-handed poly-proline helix.
Ligands for SH3 domains have been found to bind in two different orientations; either amino- terminal to carboxy-terminal, called class I ligands, or carboxy-terminal to amino-terminal, called class II ligands (Pawson, 1995). The typical sequence differs somewhat between these two classes, and although an SH3 domain can interact with both types of ligands, it normally binds only one class of motifs with high affinity, which may be functionally important in the spatial organization of signaling complexes. In addition to mediating binding to other proteins, SH3 domains are also implicated in intramolecular interactions, as exemplified by the Src SH3 domain, which is involved in repression of the catalytic activity of the kinase by interacting with sequences in the catalytic domain and linker region (Xu et al., 1997).
Examples of PDGF receptor binding molecules containing SH3 domains include Grb2, Src, Crk, p85 of PI3-K and PLC-γ.
Other protein domains also recognize proline-rich sequences, including the WW domain.
The WW domain spans about 40 amino acids and contains two highly conserved tryptophans, hence its name. It binds to proline-rich sequences within different consensus motifs, including PPXY, PPLP and PPGM (Songyang, 1999). Some WW domains can interact with phosphoserine/phosphothreonine motifs as well (Lu et al., 1999). WW domains have been found in a variety of proteins, such as formin-binding proteins (FBPs), Yes-associated protein YAP, the ubiquitin ligase Nedd4 and dystrophin. Interestingly, the juxtamembrane region of type III receptor tyrosine kinases, including the PDGF receptors, has been shown to contain a putative WW domain and indeed, the juxtamembrane region of the PDGF β- receptor was able to interact with peptides containing the PPXY consensus sequence (Irusta and DiMaio, 1998). An intriguing possibility is that phosphorylation of the two known juxtamembrane autophosphorylation sites in the PDGF receptors could regulate the binding of proteins to the WW domain.
PDZ domains are ~90 amino acids long conserved motifs originally identified in the proteins of PSD-95, Dlg-A and ZO-1. Many PDZ domains contain a relatively conserved GLGF element, and the domain has also been called GLGF repeat (and DHR). The main targets of PDZ domains are the four or five most carboxy-terminal amino acid residues of target proteins. Early studies identified a C-terminal E(S/T)XV consensus motif on subunits of NMDA and Shaker-type K+ channels, but the specificities of PDZ domains have turned out to be diverse. The only conserved motif for binding is a hydrophobic residue (usually valine, leucine or isoleucine) at the carboxyl terminus (Songyang et al., 1997). The specificity in the interaction is likely to be determined by the other residues in the consensus motif. PDZ domains often prefer a serine, threonine or tyrosine residue at the –2 position, making it possible for phosporylation events to modulate the binding (Cohen et al., 1996). The picture is complicated by reports of PDZ domains binding to internal sequences. For instance, the third PDZ domain of InaD, which mediates light response in the Drosophila eye, binds to a sequence in the Ca2+ channel protein TRP not involving carboxy-terminal residues. Moreover, homotypic interactions between PDZ domains engage internal sequences (Songyang, 1999).
Many PDZ domain-containing proteins contain multiple copies of this module. If the PDZ domains have similar binding specificities, the protein can bind several copies of the target and promote aggregation. On the other hand, proteins that contain PDZ domains with
p120RasGAP SH2 SH3 SH2 PH GAP
Grb2 SH3 SH2 SH3
Shc PTB SH2
c-Src SH3 SH2 kinase
p85 PI3-K SH3 SH2 SH2
PLC-γγγγ1 PH PLC P H
PTPase
SH2 SH2 SH3 PLC
SHP-2 SH2 SH2
LMW-PTP PTPase
c-Crk SH2 SH3 SH3
Stat DNA binding
domain SH3 SH2
SH2
SH3
PH
PTB SH2 domain
SH3 domain
PH domain
PTB domain
