The developmait o f a complex multi-cellular organism requires segregation of function; i.e embryonic cells have to differentiate into various tissue types and organ systems. Intercellular communication plays an important role in the reception and transduction o f extrinsic signals wfiich induce the e^q^ression o f intrinsic effector molecules. Since the hydrophobic lipid bi-layer of the plasma membrane delimits and establishes the cell as an autonomous unit within the aqueous environment, an elabomte and specific intercellular communication network is required for the transduction o f the signal across the otherwise impervious plasma membrane (Alberts et al., 1983).
The evolution of multi-cellular eukaryotic organisms have accompanied the parallel
evolution and conservation o f a class o f genes, the proto-oncogenes, involved in intercellular communication (Snyder, 1985; Yardenand Ullrich, 1988). Proto-oncogenes encode for proteins involved at every level of the signal transduction cascade, including the extrinsic signalling molecules (eg. growth and differentiation factors like FGF, EOF, NGF etc.) and components of the intracellular signal transduction. The intracellular signaling cascade comprise of specific cell surface signal transducing receptor molecules, a common pool of cytosolic second-messengers that relay the signal to the target effector molecules, the nuclear transcription factors (Hill and Treisman, 1995) (Fig 1.1 (A) & (B)). Activation of transcription factors initiates the appropriate cellular response to the external signal by trans-activation o f specific target genes within the responding cell. This signal transduction pathway functions to regulate cell proliferation, differentiation and survival (Zelenka, 1990).
Normal function o f proto-oncogenes in intercellular communication was realised from the studies on the role o f transforming genes (oncogenes) in cell proliferation and malignancy (Cooper, 1982; Watson et al., 1987). The subversion of the normal signal transduction pathway by gain-of-function mutations or Retro-viral or re-combination mediated gain-of-function translocation of proto-oncogenes cause oncogenic transformation by constitutive activation of the signaling cascade (Rabbitts, 1994; Baserga, 1994).
Relaying o f the signal across the cytosol involves multiple interconnecting second- messenger networks activated by the binding of the ligand to its cognate receptor (Fig 1.1 & 1.4). Receptor activation directly or indirectly causes alterations in intracellular cyclic nucleotide (cAMP & cGMP) turn-over catalyzed by mono-nucleotide cyclases (Majerus et al., 1990), phosphatidylinositol (PI) turn-over and phosphorylation catalyzed by phospholipase O' and PI-3-kinase respectively (Hunter, 1995; Chant and Stowers, 1995), changes in Ras-GDP/Ras-GTP ratio catalyzed GTPase-activating protein (GAP) and modified by GDP-exchange adaptor molecules (see below) (Hill and Treisman, 1995) and alterations in intracellular calcium homeostasis (Heldin, 1995) (Fig 1.1 (A)). These changes in turn cause the activation o f the intra-cellular serin/threonine kinases cascade (e.g. the Map kinases) leading to activation or
inhibition o f other downstream effector molecules vdiich induce cell proliferation or differentiation (Fig 1.1 (B) and Fig 1.4).
Trans-phosphorylation on free hydroxyl groups of serine, threonine and tyrosine residues o f cell-surface receptors and down-stream second messengers is the main mechanism by which the extra-cellular signal is transduced across the cell membrane and transmitted to the nucleus (Marshall, 1995). Receptor activation may alter intracellular second messenger levels directly by effecting the activity o f the catalyzing enzyme or indirectly by the phosphorylation of non-catalytic adaptor molecules. For example, EGF-receptor activation results in receptor autophosphorylation followed by the recruitment and activation of target enzymes like PI3-kinase, PLC7 or GAP as well as the Grb2/Sos adapter complex (Fig 1.4). Both GAP activity and the function of GDP-exchange adaptor molecules, like the Grb2/Sos complex, determine RAS- mediated intracellular signal transduction. Recruitment of Sos activates Ros by promoting exchange o f bound GDP for GTP. The level of GTP-bound R œ depends on the degree o f activation by Sos and the degree o f inactivation by GTPase-activating protein (GAP). GTP-bound Ras activates the MAP kinase pathway by recruiting inactive cytoplasmic Raf kinase to the membrane (Stocke et al., 1994; Leevers et al.,
1994) (Fig. 1.4).
These second-messenger cascades have evolved in early unicellular organisms (Stock, 1990; Anschutz et al., 1991; Herskowitz, 1995) and have been conserved throughout eukaryotic evolution (Hanks et al., 1988). The Ras and MAP kinase cascade have been shown to conserved in yeast (Schultz et al., 1995), Dictyosteliim (Firtel, 1991) C. elegan (Kayne and Sternberg, 1995), Drosophila (Wassarman et. al, 1995), and vertebrates (Lowy et al., 1991). It has been suggested that the evolution of these signal transduction pathways were an essential pre-requisite to multi-cellular evolution (Yarden and Ullrich, 1988). However, different receptor-ligand systems share the same second-messenger cascade in transmitting the signal (Fig. 1.1). The mechanism by which the receptor-ligand specificity is maintained during signal transduction remains unresolved (Marshall, 1995).
Fîg 1.1 Cellular mechanisms o f signal transduction o f neurotrophins.
(A) Molecular interactions o f neurotrophic receptors.
(B) Serine/threonine phosphorylation cascade in intracellular signal transduction
(A) EGF NGF Insulin
/
(B) MEK \ MAP KinaseOne class of cell-surface receptors involved with the transduction o f a specific signal from the environment to the cytosol is the Receptor Tyrosine Kinase (RTK) family o f proto-oncogenes (Hanks et a l, 1988). The c-ret proto-oncogene is a member o f this family o f signal transducing receptors.
1.2 (i) Receptor lyiDsine IGnases: Topology and Signal Tiansduction
The Receptor Tyrosine Kinase (RTK) family of proto-oncogenes represent a family o f homologous genes with similar struture and fimction (Hanks et al., 1988). The topology of this group o f conserved integral membrane proteins is characterised by the presences of a highly globular, usually cysteine-rich extracellular ligand binding receptor domain, a hydrophobic trans-membrane segment which anchors the protein within the plasma membrane and the functional cytoplasmic ligand-dependent tyrosine kinase domain (Ullrich and Schlessinger, 1990).
Receptor-ligand interaction results in the activation of the kinase domain which catalyze the transfer of y-phosphate of ATP to serine, threonine and tyrosine residues o f downstream substrates (Heldin, 1995). High affinity binding of ligand induce an allosteric conformational change which activates the kinase domain and causes dimerization with adjacait receptor molecules (Hanks, 1991; Cadena and Gill, 1992). This results in the juxtaposition of cytoplasmic kinase domains leading to trans phosphorylation o f receptors and downstream second messengers (Ullrich and
Schlessinger, 1990) (Fig. 1.1).
The interaction of the receptor tyrosine kinase with the intracellular substrates occurs by association at structurally conserved regulatory domains o f approximately 1 0 0 amino acids (Marengere et al., 1990). These conserved structural motifs, referred to as Src-homology (SH2 and SH3) and pleckstrin homology (PH) domains, form domains which recognize peptide motifs bearing phosphotyrosine residues (Pawson, 1995). 1Mutational, analyis o f the C. elegan Let23 RTK locus have shown that SH2 domain o f Grb-2 recognises the peptide motif, Tyr-X-Asn where X represents any amino acid (Kayne and Sternberg, 1995). Songyang et al. (1995) recently investigated
the substrate specificity of tyrosine kinases by using a degenerate peptide library to identify the optimal peptide substrates. Different tyrosine kinases were in general found to have unique optimal peptide substrates. However, wild type RET protein was shown to preferentially phosphorylate the optimal peptide of the EGFR, and the MENIIB mutant allele of c-RET (section 1.4) was shown to have altered substrate specificitv. The MENIIB allele of c-RET phosphorylated the optimal peptide substrates
I non-receptor tyrosine kinases
o f the , Src and Abl (Zhou et al., 1995).
1.2 (ii) Role of Receptor Tÿmsine Knases in Development
During early embryogenesis short range autocrine and paracrine cellular communication networks are important in establishing structure and function. Long range endocrine communications become established during organogenesis for the homeostatic maintenance of the established order. Genetic e^erim ents performed in vertebrates and invertebrates provide compelling evidence to suggest RTKs function as specific trans-membrane signal transducers (Hunter, 1990; Pawson and Bernstein, 1990).
1.2 (ii) (a) Invertebrates
Most extensive genetic analysis of RTK function have been performed in Drosophila Many o f the genes encoding RTKs in Drosophila have been identified and isolated from saturation mutagenesis protocols by screening for the genetic loci responsible for aberrant developmental phenotypes (Reviewed by Pawson and Bernstein, 1990).
Several well characterised Drosophila mutant alleles are encoded by various members o f the RTK family (Shilo, 1992). The early embryonic lethal alleles torso and torpedo are encoded by the Drosophila homologue of the vertebrate PDGF (Shilo, 1992) and EGF receptors respectively (Shilo and Raz, 1991). Torso and torpedo are maternally imprinted loci which determine embryonic polarity. The torso phenotype is characterised by the absence of anterior and posterior terminal structures, the acorn and telson. The torpedo phenotype is characterised by the absence of dorsal structures.
Torso and torpedo fimction independently of one another (Shilo, 1992).
The maternal cues which determine the position of terminal structures and the dorsal- ventral axis is generated during oogenesis by paracrine communications between the oocyte and its surrounding follicle cells (Shilo, 1992). The torso protein, the transcript o f which is uniformly distributed in the syncytial blastoderm, is thouÿit to sequester and transduce a signal from its spatially restricted cognate ligand (possible encoded by the torso-like locus) which is synthesised at the presumptive anterior and posterior pole o f the embiyo by the follicle cells.
Conversely, torpedo, an allele of the Drosophila EGF receptor (DER) is expressed by follicle cells and is thought to transduce a signal from an unidentified dorsally restricted signal from the oocyte (Shilo, 1992). The resulting reciprocal interaction between the oocyte and follicle cells establish the dorsal-ventral axis o f the embryo. In the absence of this reciprocal interaction development follows the default pathway with the formation of ventral structures.
Loss-of-frmction mutations in Drosophila RTKs which effect zygotic maturation and organogenesis have also been identified. The fcdnt little ball (fib) phenotype which is characterised by rounded cuticle and severe collapse o f CNS development result from null or severe alleles o f the DER locus. The temporal requirement of DER for the development of Drosophila CNS have been analyzed by studying the ability of temperature sensitive DER alleles to rescue the CNS phenotype (Raz and Shilo, 1992). Abnormal development of the CNS can be prevented by expressing DER early in the ectoderm (Raz and Shilo, 1992). Since DER is e?q)ressed in the ectoderm but not in neuroblasts and neurones, it is likely that DER is important for neuroblast specification (Raz et al., 1991; Raz and Shilo, 1993). DER function is also necessary for the differentiation and survival o f midline glial cells.
Ellipse, another allele o f the DER locus, has a dominant phenotype in Drosophila compound eye development. The ellipse mutation is characterised by a 1 Ox reduction in the number o f ommintidia in homozygous flies (see below). The DER receptor
function is required for generating the correct spacing between ommintidia clusters. The receptor is believed to be involved in the reception o f an inhibitory signal from the R8 photoreceptor cell which prevent the surrounding undifferentiated photoreceptors from acquiring R8 cell fate (Raz et al., 1991; Raz and Shilo, 1993).
Drosophila homologue of the vertebrate FGF receptor (DFGFR) is expressed in the glia and neural cells o f the CNS and in the developing tracheal system. Loss-of- function mutation o f this receptor results in the breathless phenotype characterised by abnormal development of the tracheal tree due to blockade in the migration o f tracheal pit cells (Glazer and Shilo, 1991).
Th\nn% Drosophila compound eye organogenesis, the sevenless mutant allele, encoded by RTK, is important for spatial organisation and neuronal commitment of the R7 photo-receptor cell. Drosophila compound eye is made up o f an array of approximately 800 structural units, the ommintida, comprise o f e i^ t photoreceptor cells (R1-R8), four lens-secreting cone cells and eight other accessory cells. In sevenless mutants, the R7 photoreceptor fails to differentiate into its normal neuronal phenotype and remain as an undifferentiated cone cell. Genetic mosaic experiments and expression of wild type sevenless using the ubiquitous inducible heat-shock protein promoter showed sevenless function to be cell-autonomous (Tomlinson et al., 1987; Tomlinson and Ready, 1987; Easier and Hafen, 1988). The isolation of the boss mutant, which has the same phenotype as sevenless, and subsequent analysis established the boss locus to encode the ligand of the sevenless receptor. The boss locus encodes for a seven trans-membrane domain protein with a long amino-terminal extracellular segment. This protein is specifically expressed in the R8 photoreceptor cell. Intercellular inductive interactions between R8 and R7 cells is th o u ^ t determine the neuronal fate o f the R7 photo-receptor cell (Easier and Hafen, 1990).
RTK homologues have also been identified in other invertebrates. The let-23 locus in the nematode C. elegans encodes the EGF receptor homologue Wiich function in cell fate determination (vulval versus hypodermal cells) in vulval development (Aroian and Sternberg, 1991; Aroian et al., 1990). The D PYK l and DPYK2 proteins are
developmentally regulated RTKs observed in the slyme mould Dictyosteoum. (Firtel, 1991).
1.2 (ii) (b) Vertebrates
Unequivocal demonstration of the involvement o f RTK family members in mammalian development emerged with the identification of the c-kit proto-oncogene as the product of the murine dominant white-spotting (W) locus, and the subsequent identification of its cognate ligand to be encoded for by the murine steel (SI) locus (Chabot et al., 1988; Geissler et al., 1988). Loss-of-function mutations o f the murine W and SI loci result in deleterious effects in haematopoiesis, gametogenesis and melanocyte differentiation. The haematopoietic deficiency induce anaemia and cause perinatal lethality of mice homozygous for the mutation. Early chimeric experiments had established that the W locus function was intrinsic and cell autonomous corresponding to a receptor, and the SI locus function was an extrinsic micro environmental signalling factor (Mayer and Green, 1968).
The trk family of RTK proto-oncogenes encode for receptors o f NGF and other neurotrophins (Bothwell, 1991; Chao et al., 1986; Jing et al., 1992). The trk family o f receptors (trk A, B & C) and their cognate ligands (NGF, BDNF & NT3) are expressed predominantly in the developing nervous system. This family o f receptors may function in the transduction of differentiation and/or survival signals from the micro-environment of developing neurones. Consistent with this hypothesis, lose-of- function mutations, generated by homologous recombination in embiyonic stem cells, in the murine trk receptors and their cognate ligands results in the ablation of sub-sets o f neurones of the CNS and PNS (Klein et al., 1993; Snider, 1994).
The molecular-genetic analysis of other members of the RTK family o f genes have not been analyzed to the same extend as the c-kit locus or the invertebrate RTKs. This may be because mutations in other RTK family members may not penetrate into the population, i.e they may be embryonic lethal. However, the spatial expression profile o f many RTKs during embryogenesis is providing compelling evidence to suggest that
vertebrate RTKs have similar fimction to the invertebrates homologues; i.e critical fimction in cell fate determination, differentiation and survival (Motro et al., 1991).
Members o f the polypeptide growth factor family of genes encode putative ligands for RKTs. Many members of this family of proteins are important signalling molecules which fimction as mitogens and morphogens during development. FGF and FGF- related growth factors and activin, inhibin and other members o f the TGF-|3 family of growth factors are involved in mesoderm induction and in establishing the A-P axis o f the embiyo (reviewed by Summerbell et al., 1991 and section 1.1). The cognate receptors of many o f these important ligands remains to be isolated. Recently, the egression of the murine FGF receptors fig and bek have been analyzed; their expression profile imply possible function in gastrulation and in epithelial- mesenchymal interactions during organogenesis (Orr-Urtreger et al., 1991).