Growth Factor Receptors:Role in Normal Mitogenic Signalling and Oncogenesis
A. Ullrich 1 and J. Schlessinger 2    Hämatol. Bluttransf. Vol35

1 Max-Planck-lnstitut für Biochemie, Am Klopferspitz 18A, 8033 Martinsricd, FRO.
2 Department of Pharmacology, New York University Medical Center, 550 First Avenue, New York, NY 10016, USA.

Growth factors, differentiation factors, and polypeptide hormones are crucial components of the regulatory system that coordinates development of multicellular organisms. Many of these factors mediate their pleiotropic actions by binding to and activating cell surface receptors with an intrinsic protein tyrosine kinase (PTK) activity. Figure 1 presents a schematic representation of the known growth factor receptors that bear PTK activity. Growth factor receptors with PTK activity, or receptor tyrosine kinases (R TKs), have a similar molecular topology. All possess a large, glycosylated, extracellular, ligand-binding domain, a single hydrophobic transmembrane region, and a cytoplasmic domain which contains a PTK catalytic domain (Hanks et al., 1988; Yarden and Ullrich 1988, Schlessinger 1988; Williams 1989). Primary sequence homology and distinct structural characteristics of different R TKs allow the classification of these receptors into subclasses (Fig.l). The structural features characteristic of the four subclasses include two cysteine-rich repeat sequences in the extracellular domain of monomeric subclass I receptors, disulfide-linked heterotetrameric alfa2 ß2 structures with similar cysteine-rich sequences in subclass II R TKs, and five or three immunoglobulin-like repeats in the extracellular domains of subclass III and IV RTKs, respectively. The tyrosine kinase domain of the latter is interrupted by hydrophilic insertion sequences of varying length. The availability of R TK cDNA clones has made it possible to initiate detailed structure-function analyses of the mechanisms of action of R TK family members. Numerous mutants of insulin, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor 1 (IGF-l), colony-stimulating factor 1 (CSF-l), and other receptors have been characterized in regard to their biological and biochemical properties. This has led to the establishment of a receptor domain function map and model for RTK-mediated signal generation (Fig. 2). Ligand binding to the extracellular domain of the receptor results in conformational change and subsequent oligomerization [Schlessinger 1988]. Receptor oligomerization is a universal phenomenon among growth factor receptors. It has been detected in living cells, in isolated membranes, and in preparations of solubilized and purified receptors [Schlessinger 1986; Yarden and Schlessinger 1985, 1987 a, b; Cochet et al., 1988]. It may be induced by either monomeric ligands, such as EGF, which cause receptor oligomerization by inducing conformational changes [Greenfield et al. 1989] resulting in receptor-receptor interactions [Lax et al. 1990] or by bivalent ligands, such as PDGF and CSF-1 , which mediate dimerization of neighboring rcceptors [Seifert et al. 1989; Heldin et al. 1989; Hammacher et al. 1989] .Oligomerized growth factor receptors possess elevated PTK activity [Yarden and Schlessinger 1987 a, b; Boni-Schnetzler and Pilch 1987], which leads to phosphorylation of tyrosine residues of the receptor polypeptide chain and of cellular substrates.

Fig. 1. Schematic representation of receptor tyrosine kinase subclasses. For details, see Ullrich and Schlessinger (1990)

Fig. 2. Proposed structure-function topology of the EGF receptor. Subdomains II and IV (stippled) represent the cysteine-rich regions of the extracellular domain. Most of the structural determinants that define EGF binding affinity are proposed to be located in the cleft Signal regulation formed by subdomains I and III. The symbols S and R within the PTK domain represent proposed interaction sites for substrates and regulatory factors [Ullrich and Schlessinger, 1990]

Receptor phosphorylation releases an internal constraint by stabilizing a conformation that is competent to interact with and phosphorylate cellular substrates [Honegger et al. 1988a,b]. The recent observation that phosphorylation of EG F and insulin receptors can occur by intermolecular cross-phosphorylation both in vitro and in living cells [Honegger et al. 1989, 1990; Ballotti et al. 1989; Lammers et al. 1990] further supports the importance of receptor oligomerization in the process of receptor activation. The chain of events that is initiated by tyrosine phosphorylation of cellular substrates is still poorly understood. Several R TK substates of potential biological importance have recently been identified (Figure 3). Both PDGF and EGF can induce tyrosine phosphorylation of phospholipase Cy(PLC-y) in vitro and in living cells [Margolis et al. 1989; Meisenhelder et al. 1989; Wahl et al. 1989]. In addition, PLC-y was observed to associate with the activated receptor kinases in a ligand- and kinase-dependent manner [Margolis et al. 1989, 1990a; Kumjian et al. 1989]. However, growth factor-induced inositol triphosphate (IP 3) generation appears not to be the

Fig. 3. Receptor-mediated multiple signalling pathways. Direct phosphorylation (black dots on symbols) of substrates, PLC-y, PtdIns-3 K, GAP, and raf leads to secondary events, including enzymatic activation and metabolite formation (DAG, IP3, PtdIns(3)P), activation of enzymatic functions by association, and Thr/Ser phosphorylation (white dot on symbol) of substrates [Ullrich and Schlessinger, 1990 ]PtdIns-3K: phosphatidylinositol 3-kinase; GAP: GTPase-activating protein; PtdIns(3)P: phosphatidylinositol 3-phosphate

sole mechanism leading to the initiation of DNA synthesis [Downing et al. 1989], which is compatible with the notion that the phosphatidylinositol (PI) signalling pathway does not play an essential role in the mitogenic response [Lopez-Rivas et al. 1987; L' Allemain et al. 1989; Margolis et al. 199Ob]. Other RTK substrates that have recently been identified include PI kinase and the ras binding protein GAP [Kaplan et al. 1987; Varticovski et al. 1989; Molloy et al. 1989] (Fig. 3). Similarly, it has been suggested that the c-raf protooncogene product becomes phosphorylated in response to PDGF receptor activation [Morrison et al. 1989]. Intriguingly, all proteins identified thus far as R TK targets are either components of second messenger pathways, protooncogene products, or factors that regulate the activity of protooncogene products. The importance of allosteric regulation of receptor activation and signal transduction is further emphasized by the fact that a large variety of structural alterations found in RTK-derived oncogene products lead to constitutive kinase activation and, consequently, subversion of molecular control mechanisms and alteration of receptor signals. Thus, transforming R TK derivatives serve as valuable model systems not only for studying the mechanisms of oncogenesis but also for the analysis of normal structurefunction relationships for these signaltransmitter molecules. Constitutive activation of R TK signalling functions can be achieved in a number of ways. For example, in the cases of v-erb-B and v-kit, deletion of the extracellular binding domain eliminates the negative control that this structure normally exerts on the cytoplasmic domain. Even point mutations within the extracellular domain can lead to intracellular activation, as in

Fig. 4. Transformation by receptor amplification. Schematic representation of proposed transformation model by autocrine stimulation of overexpressed receptor tyrosine kinases. Ligand (black dots) is activating receptors in the plasma membrane of a tumor cell, resulting in an amplified transforming signal

the case of v-fms mutations at residues 301 and 374 [Woolford et al. 1988; Roussel et al. 1988] (Fig. 4). These mutations appear to induce and stabilize a conformational change equivalent to that triggered by ligand binding and possibly dimerization. Another dramatic effect of a single point mutation is exemplified by the Val/Glu conversion in the neu transmembrane domain [Bargmann et al. 1986], which suggests that this part of the putative receptor is involved in an overall conformational alteration that occurs upon interaction with the yet unidentified ligand. In this case, the transmembrane mutation results in constitutive receptor oligomerization [Weiner et al. 1989]. Another type of structural alteration has been identified in the EGF receptor/erb-B system and involves mutations in the PTK core region [Massoglia et al. 1990]. Despite the presence of an intact extracellular domain, these mutations render the EGF receptor competent for mitogenic and transforming signalling without autophosphorylation. RTK-derived oncogenes possess other structural lesions such as cytoplasmic point mutations, deletions, and C-terminal truncations which appear to enhance and modulate the transforming signal [Khazaie et al. 1988; Woolford et al. 1988]. For human cancer, activating RTK mutations appear to be of minor importance. The most common cellular lesion found in human cancers involves autocrine activation in conjunction with receptor overexpression (Fig. 4). Many tumors and tumor cell lines have been found to coexpress growth factors and their receptors, including TGF-alfa, PDGF- A, PDGF-B, acidic fibroblast growth

Fig. 5. HER 2/neu gene amplification in mammary carcinoma. Southern blot hybridization analysis of chromosomal DNA from primary mammary carcinoma tumors [Slamon et al. 1987]

Fig. 6 a, b. Cell transformation by EG F receptor overexpression. NIH-3T3 cell lines HER-A and HER-B overexpressing the human EGF receptor (a) were stimulating with EGF or TGF-alfa and tested for their ability to grow in soft agar (b)

factor (FGF), basic FGF, and their specific R TKs. Thus, autocrine receptor activation represents yet another scenario of subversion of normal growth control. F or mammary and ovarian carcinoma, extensive studies have demonstrated a direct correlation between the extent of overexpression of p 185HER 2/neu and a patient's prognosis, a result which strongly suggests a critical role for this EGF receptor-like RTK in tumor progression and perhaps even tumor initiation [Slamon et al. 1989] (Fig. 5). This possibility is further supported by efficient induction of mammary carcinoma in mice by an activated neu gene product [Muller et al. 1988] and transformation of NIH-3 T3 cells by overexpression of un altered p 185HER 2/neu [Hudziak et al. 1987]. Analogous experiments with the EGF receptor indicated that autocrine stimulation of the overexpressed receptor was essential to achieve a transforming effect [Oi Fiore et al. 1987; Velu et al. 1987; Riedel et al. 1988] (Fig. 6). On the basis of these findings, strategies involving antireceptor antibodies were designed for the treatment of mammary and ovarian carcinoma. Monoclonal antibodies, such as the antiHER2/neu antibody 405, are able to interfere with autocrine activation of the receptor, which results in inhibition of tumor cell growth in tissue culture and nude mouse models (Ullrich et al., unpublished). In principle, every receptor with PTK activity has oncogenic potential. One can anticipate that many more types of activating mutations, as well as specific instances of R TK overexpression, will be detected in animal and human tumors. The molecular identification and characterization of these mutants will not only provide important insights into fundamental mechanisms underlying receptor activation and normal growth control, but may also enhance our understanding of oncogenesis and open new avenues for diagnosis and therapy.


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