Epidermal growth factor and transforming growth factor α

2 Epidermal growth factor and transforming growth factor ex C. A. BROWNE

Epidermal growth factor (EGF) currently represents an enigma among tissue growth factors. Although its activity was first described almost 30 years ago (Cohen, 1962), EGF has been readily available via a simple isolation procedure for nearly 20 years (Savage and Cohen, 1972), and has been a factor whose composition and amino acid sequence have been known for nearly as long (Savage et aI, 1972, 1973), it remains a potent substance whose true physiological roles remain unclear, whose pathophysiology can only be guessed at, and whose pharmacological potential has only recently been recognized and exploited . Despite the fact that we now have an extensive 'phenomenology' relating to EGF in the scientific literature, no one can say explicitly and precisely what the key biological role(s) of EGF are. In part this may result from the easy and wide availability of EGF, which has permitted many thousands of studies of the effects of EGF on a variety of tissues and cells in vitro to be undertaken. Thus, we have an almost bewildering plethora of observations of activities and actions of EGF on a wide range of tissues, taken at every stage of life from the preimplantation embryo to the aged and elderly organism . In face of this apparently broad range of EGF actions, it is perhaps not surprising that a clearly focused picture of the place of EGF in nature cannot be simply painted. The picture is further complicated by the recognition that EGF is part of an extended family of growth factors . This includes the closely related transforming growth factor ex (TGF-ex) (Derynck et al , 1984; Marquardt et aI, 1984) and the more recently recognized factor amphiregulin (Shoyab et aI, 1988, 1989), both of which are now established as small, soluble growth factors capable of being produced by human tissues. There arc also now known to be a series of viral growth factors which display considerable homology to EGF. These include vaccinia virus growth factor (Blomquist et al, 1984; Brown et aI, 1985; Reisner, 1985; Stroobant et al, 1985), Shope fibroma virus growth factor (Chang et al, 1987; Ye et al, 1988) and myxoma virus growth factor (Porter and Archard, 1987). These three poxvirus growth factors display 25-30% homology with EGF. It is now also clear that the EGF structural motif occurs in a wide range of proteins which have not been formally classified as growth factors. These include the soluble proteins tissue plasminogen activator (Pennica et al, 1983), the blood clotting factors 553 Bailliere's Clinical Endocrinology allCl Metabolism-« VoL 5. No .4 . December 1991 Copyright© 1991. by BaillicreTindall ISBN 0-7020-1491-5 All rights of reproduction in any form reserved

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IX, X and XII, protein C and protein S (Doolittle et ai, 1984; Cool et al, 1985), the structural proteins cntactin (Durkin et al, 1988), a major sulphoglycoprotein of the basement membrane, and the core proteins from cartilage-specific proteoglycan (Tanaka et al, 1988; Baldwin et al, 1989) and from human fibroblast proteoglycan (Krusius et al, 1987). There are also EGF-like domains in proteins found in a wide variety of species including Plasmodium [alciparum, the malaria parasite (Kaslow et al, 1988), Strongyllocentrotus purpuratus, the sea urchin (Hursh et ai, 1987), Caenorhabditis elegans, the nematode (Greenwald, 1985), and Drosophila melanogaster, the fruit fly (Hartley et ai, 1987). This extraordinary array of EGF-like sequence motifs presumably represents at least a stable and useful structural element, and may also represent a universally recognizable signal structure. The relevance of these EGF-like structures to the physiology of EGF per se remains obscure. In this chapter, I will endeavour to combine the basic biochemical information that we have now secured on EGF, its receptor, and on the related family of EGF-like factors, with some indication both of the breadth of the role of EGF and some speculation as to the future direction of EGF research. I will not attempt to cover the enormous literature concerning the biological effects of EGF. Instead, I refer the reader to an excellent recent review on this subject (Fisher and Lakshmanan, 1990).

STRUCTURE OF EGF: A HISTORICAL PERSPECTIVE EGF is a small, heat-stable polypeptide of between 48 and 53 amino acids (Table 1) which seems to be present in a wide range of species. The initial work on EGF all related to murine EGF (mEGF) which was found to be present in the male mouse submaxillary gland in large amounts (up to 1 mg per gram of wet weight of tissue), which facilitated its isolation. The presence of mEGF was originally detected by its ability to provoke

Table 1. Amino acid sequences of the EGF family of growth factors. The amino acid sequences of the EGF family of pcptides are aligned on the conserved cystcincs. The single letter code system has been used (IUI'AC-IUB Commission on Biochemical Nomenclature, 1968). A dot represents a conserved amino acid with respect of mouse EGF. An asterisk represents a missing or deleted amino acid. EGF sequences House Human

Rat Guinea-pig

Pig Rat TGF-Q Human TGF-Q Vaccinia virus growth foetor Amphiregulin

1 10 20 30 40 50 NSYPGCPSSYlX;YCLNGGVnUIIESLDSYTCNCVIGYSGDRCQTRDLRI.'1:ELR •• DSE •• L.H ••••• IID •••• Y•• A•• K.A•••• V•• I.E ••• Y••• K••••• •• NT ••• P••••••••••••• YV •• V.R. V••••••• I .E ••• 11 ••••••• u QDA •••• P.11 ••••• 11 •••••••••• NT .A ••••••• V.E •• UiQ•• DL •• " ••• SE•• P.11 ••••• 11 ••••• Y•• AV ••• A•••• F•• V.E ••• H••• K••••• VVSIIFNK •• D.IITQ •• FH'.T .RFL\'QEEKPA. V.HS•• V.V•• EIIA •• LA VVSFH~D •• D.HTQF .m'. T.RFLVQE.KPA. V.115 •• V.A•• EIIA •• LA DIPAIRL.GPEGlX; •• L.· .D. IIIARDIlX;:W •R.511 •• T. I •• QHVV. VATQR RKKKNP .NAEFQ~F. III' .E.KY •• H.EAV •• K.QQE.F.E. .GEK

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premature eyelid opening and tooth eruption when injected into neonatal mice (Cohen, 1962). In this publication, Cohen presented an amino acid composition for his 'tooth-lid factor', as EGF was then known, and noted correctly the absence of phenylalanine and lysine from the preparation, whose molecular weight he estimated to be 14638 from the composition. Cohen coined the name 'epidermal growth-stimulating protein' in 1964, when he reported that his factor caused a general increase in the thickness of the epidermis and an acceleration of epidermal keratinization (Cohen, 1964). The name 'epidermal growth factor' first appeared in 1965, when Cohen demonstrated that his mEGF was also active in promoting epidermal proliferation and keratinization in organ cultures of chick embryonic skin (Cohen, 1965), thus extending the application of mEGF activity into embryonic life, and to a non-mammalian species. The improvement of the isolation procedure (Savage and Cohen, 1972) produced sufficient mEGF of high purity that the primary sequence could be determined (Savage et ai, 1972). mEGF was found to comprise 53 amino acids and to contain six half cystines. The calculated molecular weight was 6045, and it was confirmed that mEGF lacked phenylalanine and lysine, as well as alanine. A variant (EGF-2) form of mEGFwas also described (Savage and Cohen, 1972) which lacked the C-terminal Leu-Arg dipeptide but which was fully biologically active. The three disulphide bonds within EGF were assigned as Cys6Cys20, C ys14_Cys31 and C ys34_Cys42 (Savage et ai, 1973). All three of these bonds must be intact for full biological potency of EGF (Taylor et ai, 1972). A further derivative (EGF-5) of mEGF which was biologically active was prepared by tryptic cleavage of the Arg 48_Trp49 bond to yield [1-48]mEGF (Savage et ai, 1972). Much later, other variant forms of mEGF were described, including a and ~ forms of EGF (Burgess et ai, 1982), where EGF-a is intact [1-53]mEGF and EGF-~ is the N-terminally truncated [2-53]mEGF. The same group described three further variants of EGF (EGF-al, -a2, -(3) which could be resolved by isoelectric focusing and ion exchange chromatography (Burgess et ai, 1983). The isoelectric points reported for these forms were 4.6, 4.3 and 4.1 EGF-al was the predominant form and is thought to be the 'native' [1-53]EGF, with a pH of 4.6. The other two forms probably represent deamidation products ofmEGF. All forms of EGF-a and EGF-~ were biologically active. At the same time that Cohen and his coworkers were isolating and characterizing mEGF, Gregory and his coworkers were investigating a substance that could be isolated from the urine of pregnant women. Gregory called his substance 'urogastrone' from its ability to inhibit gastric juice secretion. When the sequence of urogastrone was determined, it was noted that urogastrone bore a remarkable similarity to mEGF (Gregory, 1975) (see Table 1). Urogastrone comprised 53 amino acids, of which only 16 were different from mEGF. It is now widely accepted that urogastrone is the human homologue of mEGF. If the sequences of human EGF (hEGF) and mEGF are compared, 14 of the 16 amino acid changes could result from single base changes. This supports the idea that EGF has a highly conserved structure. Subsequently, the primary structures of EGF from the rat (rEGF), guinea-pig (gpEGF) and pig (pEGF) have been reported (Simpson

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etal, 1985; Pascall etal, 1991). The general featuresofhEGFand mEGFare retained in all of these structures. It is interesting that mEGF, hEGF and pEGF are all 53 amino acids long, whereas both rEGF (48 amino acids) and gpEGF (51 amino acids) are C-terminally truncated. Thus, gpEGF is analogous in length to the variant mEGF-2 and rEGF is analogous to the variant mEGF-5. There were also N-terminal variants reported for the rEGF sequence which was found in three truncated forms, 2-48, 3-48 and 4-48 (Simpson et aI, 1985). All of these forms were biologically active. Thus, from all of these 'naturally occurring' variant forms of EGF, we may conclude that the extreme Nand C termini are not important for biological activity , and that a core of [4-48]EGF in all species would be sufficient for biological activity. Studies with artificially gen erated [1-47]hEGF indicated that it too was equipotent with [1-53]hEGF (Hollenberg and Gregory, 1976). When we compare the known EGF primary sequences, several points can be noted (see Table 1). Firstly there is a single methionine at position 21 which is invariant in all known EGFs, and is required for biological activity. Secondly, the six cystine residues are also invariant, as indeed they are in all the EGF-related structures. Thirdly, the amino acid changes are quite widely distributed throughout the molecule. In total, there is a 50% conservation of amino acids between these sequences (24 out of 48 equivalent positions). The most highly conserved region appears to be from Ser? through to Glu 24 • Interestingly, there seems to be some strong restriction on the nature of the changes permissible in this region. For instance, position 10 is tyrosine in mEGF and rEGF, but is histidine in the other known EGF (and TGF-a) sequences, whereas position 22 is histidine in mEGF and gpEGF, but is tyrosine in hEGF, rEGF and pEGF. (Interestingly, this equivalent position is phenylalanine in TGF-a sequences and histidine in the vaccinia virus sequence.) Position 16 is asparagine or histidine in all eight sequences shown in Table 1. In other regions of the molecule, invariant amino acids also appear at positions Tyr37 , Gly39, Arg 4 1 , and Leu'". Prior to any direct three-dimensional structural information on EGF, the molecule was described as comprising three loops which were held together by the disulphide bonds. These three loops were 6-13 as the A loop, 14-31 as the B loop and 33-42 as the C loop. The N- and C-terminal regions were both regarded as being less important for activity, due to the apparent unimportance of their presence. No one has been able to produce a small peptide fragment which has full EGF-like activity. The first claim to any success in this has been by Komoriya et al (1984), who synthesized a series of peptides which were part analogues of EGF and reported that peptides which contained the [20-31]EGF sequence showed some activity, albeit at 10- 4 the potency of native [1-53]EGF. This 20-31 region is in the B loop of EGF and it was suggested that this may represent a receptor-binding dom ain for EGF. Howev er, since this fragment was only 10- 4 times the activity of the native molecule, the significance of this finding was unclear. Moreover, the sequences of EGFs are not particularly well conserved in this region (20-31 comprises five invariant amino acids in 11, including the two cysteines at 20 and 31).

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There are still no reported X-ray crystallography structures of EGF or EGF-like molecules. However, there have been several reports of nuclear magnetic resonance (NMR) structural determinations of both EGFs and TGFs. There have been solution structures defined by NMR methods for mEGF (Montelione et aI, 1986, 1987) and hEGF (Cooke et aI, 1987)and there has been a limited assessment of the rEGF structure by NMR (Mayo et aI, 1986). More recently, the hTGF-a solution structure has been independently determined by three groups (Brown et aI, 1989; Kohda et aI, 1989; Montelione et aI, 1989). All of these structures conform to the same basic pattern, which has been described as the 'mitten' model. The three disulphide bonds hold the molecule in this structure, where a major 13 sheet from the B loop area comprising a first strand from residues 19-24, then a 13 turn at residues 24-27 and then a second strand running from residues 27-32, and a minor 13 sheet (residues 37-38 with residues 44-45 with a 13 turn at residues 39-42, form the 'hand of the mitten. The N-terminal region and first part of loop A form the 'thumb'. A nice image from this is that the EGF may 'grasp' the receptor between the N-terminal region 1-6 and the outer region of the first strand of the major 13 sheet (residues 21-25). It is of note that the 'biologically active' peptide 20-31 of Komiriya et al (1984) comprises almost the whole of the major 13 sheet area. In all of the NMR studies, there was evidence for a weak interaction between the N-terminal residues and the outer section of the first strand of the major 13 sheet. All the NMR studies also have supported the notion of a 'cluster' of aromatic residues occurring in the EGF structures. In human EGF, this consists of Tyr 13 , Tyr22 and Tyr29 • In murine EGF, Tyr l O participates in this cluster instead of position 22, which is a histidine. The other invariant tyrosine in EGF is at position 37. This is involved in the stabilization of the type 1113 turn, which is the 'palm' of the 'mitten' joining together the two 13 sheet domains, by the formation of a hydrogen bond with invariant Va1 34 • The primary sequence is highly conserved in the region 31-37 which encompasses the two residues Cys3 1 and Cys33 , which are crucial in holding the two halves of the molecule together, and the type 1113 turn region. It is interesting that earlier reports on the solution structure of EGF have been vindicated by the NMR studies. Thus, Taylor reported that circular dichroism studies revealed no a helix and about 25% 13 structure in mEGF (Taylor et aI, 1972). Holladay et al (1976) also reported the presence of the 13 structure by circular dichroism, and he also reported that perturbation of residues 21 and 22 (now known to be in the major 13 sheet) reduced the amount of 13 structure. It is still difficult to reconcile the marked thermal stability of EGF with the structural information that we now possess. BIOSYNTHESIS OF EGF There were several early reports of high molecular weight forms of mEGF, which were stable between pH 5 and 8 (Taylor et aI, 1970, 1974). The nature of this 74 000 Da high molecular weight storage form of mEGF was found to be an aa1313 tetramer comprising two molecules of [1-53]EGF and two

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molecules of an argmme esterase protein. At low pH this complex dissociates to release [1-53]EGF (Taylor, 1974). It has been suggested that, since the terminal amino acid of mEGF is an arginine, the arginine esterase cleaves EGF from its biosynthetic precursor and the cleaved [1-53]EGF then binds to the enzyme which liberated it. A similar system operates for the biosynthesis of 13 nerve growth factor in the male mouse submaxillary gland (Server and Shooter, 1976). Interestingly the C-terminally truncated forms such as [1-48]mEGF and [1-51]mEGF do not bind to the arginine esterase (Server et aI, 1976). It is possible that it is the arginine esterase itself which produces the C-terminally truncated forms. Thus, the use of acidic extraction media for the isolation of EGF is preferred (Elson et aI, 1984) as this can prevent the production of these altered forms of EGF and will ensure that the high molecular weight complex is dissociated. A putative biosynthetic intermediate of EGF has been described (Frey et aI, 1979) which is of molecular weight 9000 and could be converted to [1-53]EGF by treatment with the arginine esterase. With the advent of recombinant DNA technology, the molecular biology of EGF biosynthesis has become much more clearly understood. The mEGF precursor protein was deduced from the cDNA to comprise 1217 amino acids (Scott et aI, 1983; Gray et aI, 1983) and to have a molecular weight of 128000. The first 25 amino acids from the N terminus are thought to represent the 'pre'-signal sequence. Mature [1-53]EGF is represented by residues 976-1029 of the precursor. The precursor was remarkably found to contain seven other cysteine-rich regions with a high degree (20-40%) of sequence homology to EGF (Scott et aI, 1983). Each of the EGF-Iike regions is flanked by a basic amino acid. The precursor also contains four N-glycosylation sites and several dibasic amino acid sites, thought to be important as potential sites for intracellular processing of the precursor. Interestingly, the most conserved region in the seven EGF-Iike sequences is the span equivalent to [29-37]mEGF in the 'authentic' [1-53]EGF. This region was earlier noted to be highly conserved between EGF species and to contain the crucial Cys3 1 and Cys33 residues and the type 1113 turn. The extreme C terminus of the precursor has a 20 amino acid hydrophobic region (residues 1039-1058), just down-sequence from the 'authentic' EGF sequence by ten amino acids. It has been suggested that this motif could act as a transmembrane region which serves to present the bulk of the EGF precursor as a cell membrane-bound glycoprotein with all the carbohydrate sites and EGF-Iike sites on the extracellular surface of the plasma membrane (Scott et aI, 1985). The portions of the precursor which contain the EGF-Iike sequences are found in two clusters separated by 400 amino acids. This arrangement is remarkably homologous to the low-density lipoprotein receptor (Sudhof et aI, 1985), which has one extracellular domain containing two well-separated clusters of EGF-Iike sequences. A similar precursor structure, comprising 1207amino acids, has been deduced for hEGF (Bell et al,1986). Doolittle et al (1984) pointed out some other overlooked homologies within the EGF precursor to the bovine and human factors X and IX of the blood-clotting system, and bovine protein C. Thus the EGF precursor is

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probably an ancient and early protein in evolutionary terms. It is interesting to speculate that the well-known phenomenon of interference in EGF receptor assays by some component of human serum but not plasma (Bowen-Pope and Ross, 1983) may be due to the presence of the EGF-like motifs from factor X or factor IX. It may be that the EGF-like structural motif represents a simple way of constructing a small highly stable protein structural element built around a type 1113 turn stabilized by a disulphide bond, that nature has reused in different guises to perform different functions. There is no evidence that any of the EGF-like sequences within the EGF precursor are cleaved from the precursor. There is also no evidence to suggest that any of them represent biologically active EGF. However, one very interesting possibility is that the normal expression of EGF in most tissues is to form the membrane-bound glycoprotein precursor which can perhaps act directly as a cell-to-cell growth factor while it is held within the cell membrane or, perhaps, it may be available for cleavage by extracellular proteins or elements of the extracellular matrix to liberate 'authentic' EGF locally. It may be that the male mouse submaxillary gland is anomalous because it has the arginine esterase capable of cleavage of the precursor to 'authentic' EGF, which can then be easily stored within the cell. Therefore we have the possibility of EGF acting as a 'classical' soluble growth factor, a cell membrane-bound factor, perhaps involved in cell-to-cell interactions, or cell-to-extracellular-matrix interactions, or finally even perhaps a receptor for some other factor, whose action involves the release of EGF. The precursor form of EGF has indeed been found to occur in the distal convoluted tubules of the mouse kidney (RaIl et ai, 1985), where it may act to regulate some aspect of tubular transport. Although the existence of an active form of the cell membrane-bound EGF precursor has yet to be unequivocally demonstrated, an elegant transfection experiment has demonstrated that a biologically active form of the kidney-derived EGF precursor can be expressed in artificially constructed cells (Mroczkowski et ai, 1989). These cells both produced soluble EGF and possessed a cell membrane-incorporated EGF precursor which was biologically active. A similar transfection experiment has demonstrated that the analogous TGF-a precursor can be expressed as a cell surface glycoprotein with biological activity in the absence of any further processing (Wong et ai, 1989). It is tempting to speculate that the role of the EGF precursor in kidney tubular transport may be on Na+/H+ ion exchange. One of the described early actions of EGF on cells is to alter Na+/H+ balance by causing increased sodium entry to and hydrogen ion export from cells (Rozengurt, 1983) via a mechanism which is sensitive to amiloride, a diuretic which blocks Na+/H+ exchange (Moolenaar et ai, 1982). The distal convoluted tubule is a major site of Na+/H+ exchange and it has been speculated that renal EGF may have a role in the regulation of this exchange (Scott et ai, 1985). One must also suggest that the urinary EGF found in several species might be derived by degradation of this pro-EGF membrane glycoprotein. Indeed, Hirata and Orth have reported that high molecular weight forms of EGF are found in human urine. These high molecular weight forms of hEGF did not dissociate to authentic EGF at low pH (Hirata and Orth, 1979). They

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reported that these forms of EGF were 28000 and 33 000 molecular weight glycoproteins with a pI around 3.9. It is not impossible to construe that these EGF-Iike molecules were parts of the extracellular domain of the EGF precursor, presumably comprising the authentic EGF and part of the contiguous amino region of the precursor which contains two consensus glycosylation sites and four of the seven 'EGF-like' regions. The most C-terminal of the dibasic amino acid sequences occurs proximal to this region, so that an EGF-containing glycoprotein, which lacks the putative C-terminal membrane spanning region and contains the four 'silent' EGFlike sequences, could be cleaved from the precursor. Such a molecule would have a molecular weight around 30000. It remains to be seen if this form of EGF can be positively identified. Interestingly, the large molecular weight forms of Hirata and Orth were biologically active, albeit with only 25% of the potency of authentic EGF. There is also some independent evidence that urinary EGF is renal in origin. People with kidney diseases characterized by nephron loss have low concentrations of urinary EGF (Mattila et ai, 1986) and unilateral nephrectomy in the rat reduced urinary EGF concentrations by half (Olsen et al, 1984). These ideas for a different origin for blood and urinary EGF have other support. Androgen treatment of castrated mice stimulated EGF in the submaxillary gland and the blood, but had no effect on the urinary concentration (Perheentupa et al, 1985). Conversely, thyroxine treatment increased EGF concentration in urine, but decreased it in blood (Perheentupa et al, 1984). THE EGF RECEPTOR EGF is capable of stimulation of the proliferation of many different cell types. The major requirement for responsiveness to EGF is of course possession of an EGF receptor. Most mammalian cells, with the exception ofsome of the differentiated white blood cells, do express the EGF receptor. The EGF receptor is typically present at around 1O~-105 sites per cell for most non-transformed cells (Hollenberg and Cuatrecasas, 1973) and the apparent binding constant for dissociation is typically less than 1 nxr. In transformed cells the number of receptor sites may increase to the range of 106_107 sites per cell (Haigler et ai, 1978). It has been widely demonstrated that EGF binding to its receptor causes the rapid formation of clusters of EGF-receptor complexes on the cell surface (Carpenter and Cohen, 1976; Schlessinger et al, 1978), which are highly mobile with a lateral diffusion coefficient of 5 X 10- 10 cm2/s at 23°C (Shechter et al, 1978a). The EGFreceptor complexes become 'captured' by c1athrin-coated pits, and are then internalized within 30 min. This event involves EGF degradation within the lysosomal vesicles and a form of receptor down-regulation by removal of receptor sites from the cell surface. This process of receptor-mediated endocytosis and degradation of EGF is a clear consequence of receptorligand binding, but it is not necessary for the biological activity. Thus, inhibitors of degradation .of EGF, such as leupeptin, do not inhibit the mitogenic response (Savion et al, 1980) and substances that inhibit the

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formation of the coated pit clusters actually enhance the mitogenic activity (Maxfield et al, 1979). It has been suggested that a prolonged (- 8 h) occupancy of the receptor is needed for the full expression of mitogenic activity (Shechter et al, 1978b) and hence 'the processes of hormone internalization, degradation and down regulation may be irrelevant to the effects of the hormone' (Shechter et al, 1978b). EGF-receptor internalization leads to the destruction of the receptor (Carpenter, 1985). This process is accelerated ten-fold by the presence of EGF. There seems to be very little receptor recycling. Since this process occurs rapidly (- 30 min), and the time for full signal transduction appears to be prolonged (-8 h), the cell must be engaged in a rapid synthesis of EGF receptor to avoid depletion at the cell surface. Biosynthetic pulse-ehase experiments seem to have established that mature receptor appears at the cell surface 2-3 h following a 15 min pulse-labelling period (Stoscheck et ai, 1985). A variety of biosynthetic studies using SDS-polyacrylamide gel electrophoresis and immunoprecipitation have established that the mature receptor is a 170000Da glycoprotein (Das et ai, 1977; Cohen et ai, 1982). An 'immature' form of the receptor has also been described. This is a 160000 molecular weight glycoprotein that can be reduced in size to 130000 Da by treatment with endoglycosidase H (Soderquist and Carpenter, 1984). Treatment of the mature 170000 Da receptor with endoglycosidase fails to reduce its mass. Biosynthetic studies performed in the presence of tunicamycin, an inhibitor of N-glycosylation, also results in a 130000 Da receptor protein, which is clearly the polypeptide chain component. This 130000 Da form of the receptor fails to bind EGF (Soderquist and Carpenter, 1984). One of the earliest events following EGF binding to its receptor is an increase in the phosphorylation of a number of key intracellular regulatory proteins (Cohen et ai, 1982), including several phospholipases (Margolis et ai, 1989), and protein kinases and phosphatases (Yang et ai, 1989). The EGF receptor has been demonstrated to be a potent protein kinase which can be autophosphorylated at tyrosine residues (Ushiro and Cohen, 1980; Hunter and Cooper, 1981) as well as being phosphorylated at serine and threonine residues by protein kinase C (Hunter et ai, 1984). There seem to be at least three autophosphorylation sites close to the C terminus of the receptor (Downward et ai, 1984a). The structure of the EGF receptor has been deduced from cDNA studies and limited peptide mapping (Downward et ai, 1984b; Ullrich et ai, 1984). The receptor is a single-chain glycoprotein of 1186 amino acids, which comprises an extracellular EGF-binding domain (the N-terminal 620 amino acids, a transmembrane domain of 24 amino acids, an intracellular space of 50 amino acids, an intracellular protein kinase domain of250 amino acids and an extreme C-terminal domain of250 amino acids which contains the autophosphorylation sites (Hunter et ai, 1984; Ullrich et ai, 1984). The carbohydrate is all contained within the extracellular EGF-binding domain. The existence of 'active' fragments of the EGF receptor has been reported. A soluble 105 kDa protein can be produced by A-431 cells in culture. This binds EGF and has all of the characteristics of the EGFbinding domain of the EGF receptor (Weber et ai, 1984). There is evidence

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that this EGF 'binding protein' was biosynthesized independently from an alternative mRNA rather than being proteolytically cleaved from the cell membrane-bound receptor (Lin et ai, 1984; Weber et ai, 1984). A 42 kDa protein has been proteolytically cleaved from the EGF receptor in vitro, and has been shown to retain protein kinase activity, even in the absence of the EGF-binding domain and the autophosphorylation domain (Basu et ai, 1984). The most remarkable finding from these studies on the EGF receptor (Downward et ai, 1984b) was the recognition that the receptor has startling homology with the erb-B viral oncogene product that had been deduced previously (Yamamoto et ai, 1983). Six peptides within the EGF receptor are highly conserved (74 out of 83 amino acids) in the erb-B protein. In essence the erb-B protein resembles the EGF receptor without the EGFbinding domain. In other words the erb-B product and the EGF receptor have very similar transmembrane, tyrosine kinase and autophosphorylation domains. Thus the oncogenic activity of the erb-B protein can be postulated to be expressed as a constitutive, permanently activated, unregulatable EGFreceptor. Both the EGFreceptor gene and theerb-B gene are found on the long arm of human chromosome 7, and it has been suggested that the EGF receptor gene has given rise to the erb-B oncogene. EGF SIGNAL TRANSDUCTION

EGF stimulates cell proliferation in a wide range of cells (Fisher and Lakshmanan, 1990). It does so by binding to the EGF receptor and activating intracellular processes, primarily as a consequence of the tyrosine kinase activity of the receptor (Hunter and Cooper, 1981). Furthermore, the phosphorylation of phospholipase C-lI (Margolis et aI, 1989) leads to the activation of protein kinase C (Wahl and Carpenter, 1988) which causes the activation of the inositol phosphate pathway. The primary mitogenic response to EGF seems not to involve either cyclic nucleotides or G proteins, although there is some evidence which suggests that EGF can induce granulosa cell differentiation via a mechanism involving cAMP (Knecht and Catt, 1983). Since EGF is internalized into the cell via the receptor-mediated mechanism outlined above, the possibility exists for a direct involvement of either intracellular EGF or EGF receptor in signal transduction, if one presupposes that not all of the internalized EGF and receptor are rapidly degraded within the lysosomes. There is some evidence to support these ideas, in that direct interactions of EGF with chromatin (Rakowicz-Szulczynska et al, 1989) and of EGF receptor with supercoiled DNA (Mroczkowski et al, 1984) have been reported. TRANSFORMING GROWTH FACTOR

(X

Within the limited scope of this chapter, I can only afford a brief summary of TGF-a. Our present knowledge is probably best described by noting that TGF-a has a similar primary structure to EGF (Derynck et aI, 1984;

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Marquardt et ai, 1984), has a very similar solution structure to EGF (Brown et ai, 1989; Kohda et ai, 1989; Montelione et ai, 1989), is produced from a similar precursor to EGF (Derynck et ai, 1984; Lee et ai, 1985) and that TGF-o: can bind to and activate the EGF receptor in an analogous manner to EGF (Pike et ai, 1982). TGF-o: is produced by normal tissues such as pituitary (Samsoondar et ai, 1986) and decidua (Han et ai, 1987), but its production is enhanced by cells which have become transformed into tumour cells (Todaro et ai, 1980). There is evidence to suggest that a cell membrane-incorporated form of the TGF-o: precursor can be biologically active (Wong et ai, 1989). Other than its role in tumour cell proliferation, the most important role of TGF-o: is probably in prenatal development. EGF receptor is expressed in a wide range of embryonic (Adamson et ai, 1981) and fetal (Nexo et ai, 1980) tissues, from as early as the preimplantation stages (Wood and Kaye, 1989). EGF-like activity has been identified in fetal and embryonic tissues (Nexo et ai, 1980; Mesiano et ai, 1985) and EGF has been shown to have profound effects on the developing fetus (Thorburn et ai, 1981). However, EGF precursor mRNA does not seem to be expressed in significant levels during fetal development at times when the EGF receptor is clearly present (Freemark and Comer, 1987; Snead et ai, 1989), whereas there is evidence that TGF-o: is expressed during development (Lee et ai, 1985b; Twardzik, 1985; Han et ai, 1987). Thus the current speculation is that TGF-o: is the ligand for the EGF receptor during fetal and embryonic life, that EGF is the normal ligand from the neonatal period into adult life and that TGF-o: expression may become predominant again if and when cells become tumorous. However, this somewhat simplistic model should not necessarily preclude a role for TGF-o: in normal neonatal development (Tam, 1985). SUMMARY

I have attempted here to outline the basic biochemical knowledge that we have now secured on the EGF family of proteins. In the future we will learn much more about the differential role of EGF versus TGF-o:, about the physiological significance of amphiregulin, the newest member of this family, and about the roles of TGF-o: and amphiregulin in cancer. Many questions remain. What is the importance of these factors in embryogenesis and fetal development? Is there an involvement of the EGF-Iike domains of extracellular proteins in cell-to-extracellular-matrix interactions? Do these extracellular matrix EGF-like entities function in a similar manner to fibroblast growth factor in cell growth and in mediating the relationship of cells to the extracellular matrix? What is the significance of cell membrane-bound forms of EGF and TGF-o: as potential cell-to-cell contact regulators? What is the role of the viral EGF-Iike proteins in the viral infective and transforming process? These and other questions will be addressed in the next decade. The key question has already been well stated: 'what is the normal physiological role of EGF during development and homeostasis? The answers to

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these and a host of other questions must be found before we can fully comprehend this important regulatory system' (Cohen, 1987).

REFERENCES Adamson ED, Deller MJ & Warshaw JB (1981) Functional EGF receptors are present on mouse embryo tissues. Nature 291: 656-{)59. Baldwin Cf', Reginato AM & Prockop DJ (1989) A new epidermal growth factor-like domain in the human core protein for the large cartilage-specific proteoglycan. Journal of Biological Chemistry 2~: 15747-15750. Basu M, Biswas R & Das M (1984) 42,OOO-molccular weight EGF receptor has protein kinase activity. Nature 311: 477-480. Bell or, Fong NM, Stempien MM, Wormsted MA, Caput D, Ku L, Urdea MS, Rail LB & Sanchez-Pescador R (1986) Human epidermal growth factor precursor: eDNA sequence, expression in vitro and gene organization. Nucleic Acids Research 14: 8427-8446. Blomquist MC, Hunt LT & Barker WC (1984) Vaccinia virus 19 kilodalton protein: relationship to several mammalian proteins, including two growth factors. Proceedings of the National Academy of Sciences of the USA 81: 7363-7367. Bowen-Pope DF & Ross R (1983) Is epidermal growth factor present in human blood? Interference with the radioreceptor assay for epidermal growth factor. Biochemical and Biophysical Research Communications 114: 1036-1041. Brown JP, Twardzik DR, Marquardt H & Todaro GJ (1985) Vaccinia virus encodes a polypeptide homologous to epidermal growth factor and transforming growth factor. Nature 313: 491-492. Brown SC, Mueller L & Jeffs PW (1989) IH NMR assignment and secondary structural elements of human transforming growth factor alpha. Biochemistry 24: 593-599. Burgess AW, Knesel J, Sparrow LG, Nicola NA & Nice EC (1982) Two forms of murine epidermal growth factor: rapid separation by using reverse-phase high performance liquid chromatography. Proceedings of the National Academy of Sciences of the USA 79: 57535757. Burgess AW, Lloyd CJ & Nice ED (1983) Murine epidermal growth factor: heterogeneity on high resolution ion-exchange chromatography. EM BO Journal 2: 2065-2069. Carpenter G (1985) Epidermal growth factor: biology and receptor mechanism. Journal ofCell Science supplement 3: 1-9. Carpenter G & Cohen S (1976) (2SI-labelled human epidermal growth factor (hEGF): binding, internalization, and degradation in human fibroblasts. Journal ofCell Biology 71: 159-171. Chang W, Upton C, Shiu-Lok H, Purchio AF & McFadden G (1987) The genome of Shope fibroma virus, a tumorigenic poxvirus, contains a growth factor gene with sequence similarity to those encoding epidermal growth factor and transforming growth factor alpha. Molecular and Cellular Biology 7: 535-540. Cohen S (1962) Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the new-born animal. Journal of Biological Chemistry 237: 15551562. Cohen S (1964) Isolation and biological effects of an epidermal growth-stimulating protein. National Cancer Institute Monographs 13: 13-37. Cohen S (1965) The stimulation of epidermal proliferation by a specific protein (EGF). Developmental Biology 12: 394-407. Cohen S (1987) Epidermal growth factor in vitro. Cellular and Developmental Biology 23: 239-246. Cohen S, Ushiro H, Stoscheck C & Chinkers M (1982) A native 170,000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles. Journal of Biological Chemistry 257: 1523-1531. Cooke RM, Wilkinson AJ, Baron M, Pastore A, Tappin MJ, CampbelllD, Gregory H & Sheard B (1987) The solution structure of human epidermal growth factor. Nature 327: 339-341. Cool DE, Edgell CS, Louie GV. Zoller MJ, Brayer GD & MacGillivray RTA (1985)

EGF AND TGF-Ct

565

Characterization of human blood coagulation factor XII eDNA. Journal of Biological Chemistry 260: 13666-13676. Das M, Miyakawa T, Fox CF, Pruss R~l, Aharonov A & Herschman HR (1977) Specific radiolabelling of a cell surface receptor for epidermal growth factor. Proceedings of the National Academy of Sciences of the USA 74: 2790-2794. Derynck R, Roberts AV, Winkler ME, Chen EY & Goeddel DV (1984) Human transforming growth factor-a: precursor structure and expression in E. coli. Cell 38: 287-297. Doolittle RF, Feng DF & Johnson MS (1984) Computer-based characterization of epidermal growth factor precursor. Nature 307: 55&-560. Downward J, Parker P & Waterfield MD (1984a) Autophosphorylation sites on the epidermal growth factor receptor. Nature 311: 483-485. Downward J, Yarden Y, Mayes E, Scrace G, Totty N, Stockwell P, Ullrich A, Schlessinger J & Waterfield MD (1984b) Close similarity of epidermal growth factor receptor and v-erb-B oncogene protein sequences. Nature 307: 521-527. . Durkin ME, Chakravarti S, Bartos BB, Shu-Huang Liu, Friedman RL & Chung AE (1988) Amino acid sequence and domain structure of entactin. Homology with epidermal growth factor precursor and low density lipoprotein receptor. Journal of Cell Biology 108: 27492756. Elson SD, Browne CA & Thorburn GD (1984) Extraction and purification of murine epidermal growth factor. Biochemistry lnternational S: 427-435. Fisher DA & Lakshmanan J (1990) Metabolism and effects of epidermal growth factor and related growth factors in mammals. Endocrine Reviews 11: 418-442. Freemark M & Comer M (1987) Epidermal growth factor (EGF)-like transforming growth factor (TGF)-activity and EGF receptors in ovine fetal tissues: possible role for TGF in ovine fetal development. Pediatric Research 22: 609-615. Frey P, Forand R, Maciag T & Shooter EM (1979) The biosynthetic precursor of epidermal growth factor and the mechanism of its processing. Proceedings ofthe National Academy of Sciences of the USA 76: 6294-6298. Gray A, Dull TJ & Ullrich A (1983) Nucleotide sequence of epidermal growth factor eDNA predicts a 128,000 molecular weight precursor. Nature 303: 722-725. Greenwald 1 (1985) lin-l2, a nematode homeotic gene, is homologous to a set of mammalian proteins that include epidermal growth factor. Cell 43: 583-590. Gregory H (1975) Isolation and structure of urogastrone and its relationship to epidermal growth factor. Nature 275: 325-327. Haigler H, Ash JF, Singer SJ & Cohen S (1978) Visualization by fluorescence of the binding and internalization of epidermal growth factor in human carcinoma cells A-431. Proceedings of the National Academy of Sciences of the USA 75: 3317-3321. Han VKM, Hunter ES, Pratt RM, Zendegui JG & Lee DC (1987) Expression of rat transforming growth factor alpha mRNA during development occurs predominantly in the maternal decidua. Molecular and Cellular Biology 7: 2335-2343. Hartley D, Xu T & Artavanis-Tsakonas S (1987) The embryonic expression of the Notch locus of Drosophila melanogaster and the implications of point mutations in the extracellular EGF-like domain of the predicted protein. EMBO Journal 6: 3407-3417. Hirata Y & Orth DN (1979) Epidermal growth factor (urogastrone) in human fluids: size heterogeneity. Journal of Clinical Endocrinology and Metabolism 48: 673-679. Holladay LA, Savage CR, Cohen S & Puett D (1976) Conformation and unfolding thermodynamics of epidermal growth factor and derivatives. Biochemistry 15: 2624-2633. Hollenberg MD & Cuatrecasas P (1973) Epidermal growth factor: receptors in human fibroblasts and modulation of action by cholera toxin. Proceedings ofthe National Academy of Sciences of the USA 70: 2964-2968. Hollenberg MD & Gregory H (1976) Human urogastrone and mouse epidermal growth factor share a common receptor site in cultured human fibroblasts. Life Sciences 20: 267-274. HunterT & Cooper JA (1981) Epidermal growth factor induces rapid tyrosine phosphorylation of proteins in A431 human tumor cells. Cell 24: 741-752. Hunter T, Ling N & Cooper JA (1984) Protein kinase C phosphorylation of the EGF receptor at a threonine residue close to the cytoplasmic face of the plasma membrane. Nature 311: 480-483. Hursh DA, Andrews ME & Raff RA (1987) A sea urchin gene encodes a polypeptide homologous to epidermal growth factor. Science 237: 1487-1490.

566

C. A. BROWNE

IUI'AC-IUB Commission on Biochemical Nomenclature (1968) A one letter notation for amino acid sequences. Tentative rules. Iournal of Biological Chemistry 243: 3557-3559. Kaslow DC, Quackyi lA, Syin C, Raurn MG, Keister DB, Coligan Jfi, McCutchan TF & Miller LH (1988) A vaccine candidate from the sexual stage of human malaria that contains EGF-like domains. Nature 333: 74-76. Knecht M & Catt KJ (1983) Modulation of cAMP-mediated differentiation in ovarian granulosa cells by epidermal growth factor and platelet-derived growth factor. Journal of Biological Chemistry 258: 2789-279-1. Kohda D, Shimada I, Miyake T, Fuwa T & Inagaki F (1989) Polypeptide chain fold of human transforming growth factor alpha analogous to those of mouse ami human epidermal growth factors as studied by III NMR. Biochemistry 28: 953-958. Komoriya A,lIortsch M, Meyers C, Smith M, Kanety H & Schlessinger J (198-1) Biologically active synthetic fragments of epidermal growth factor: localization of a major receptor binding region. Proceedings of the National Academy of Sciences of the USA 81: 13511355. Krusius T, Gehlsen K & Ruoslahti E (1987) A fibroblast chondroitin sulphate protcoglycan core protein contains lectin-like and growth factor like sequences. Journal of Biological Chemistry 262: 13120--13125. Lee DC, Rose TM, Webb NR & Todaro GJ (1985a) Cloning and sequence analysis of a cDNA for rat transforming growth factor o , Nature 313: 489-491. Lee DC, Rochford R, Todaro GJ, Villarreul LP (1985b) Developmental expression of rat transforming growth factor alpha mRNA. Molecular and Cellular Biology 5: 3644-3652. Lin CR, Chen WS, Kruiger W, Stolarsky LS, Weber W, Evans R~I, Verma 1M. Gill GN & Rosenfeld MG (198-1) Expression cloning of human EGF receptor complementary DNA: gene amplification and three regulated messenger RNA products in A-131 cells. Science 224: 8-13-8-18. Margolis B, Rhee SG, Felder S, Mcrvic M, Lyall R, Levitzki A. Ullrich A, Zilberstcin A & Schlessinger J (1989) EGF induces phosphorylation of phospholipase C-II: a potential mechanism for EGF receptor signaling. Cel/57: 1101-1107. Marquardt J, Hunkapillcr MW, Ilood L & Todaro GJ (198-1) Rat transforming growth factor type I: structure and relation to epidermal growth factor. Science 223: 1079-1082. Mattila A-L, Perhccntupa J, I'esonen K & Viinikka L (1985) Epidermal growth factor in human urine from birth to puberty. Journal ofClinical Endocrinology and Metabolism Ski 997-1000. Mayo KII, Schau dies P, Savage CR, DcMarco A & Kapstein R (1986) Structural characterization and exposure of aromatic residues in epidermal growth factor from the rat. Biochemical Iournal Tyl: 13-18. Maxfield FR, Davies PJA, Klempner L, Willingham MC & Paston I (1979) Epidermal growth factor stimulation of DNA synthesis is potentiated by compounds that inhibit its clustering in coated pits. Proceedings ofthe National Academy ofSciences ofthe USA 76: 5731-5735. Mesiano S, Browne CA & Thorburn GD (1985) Detection of endogenous epidermal growth factor-like activity in the developing chick embryo. Developmental Biology 110: 23-28. Montelione GT, Wiithrick K, Nice EC, Burgess A W & Schcraga IIA (1986) Identification of two antiparallel beta sheet conformations in the solution structure of murine epidermal growth factor by proton nuclear magnetic resonance. Proceedings ofthe National Academy of Sciences of the US,\ 83: 8594-8598. Montelione GT, Wiithrick K, Nice EC, Burgess A W & Scherage HA (1987) Solution structure of murine epidermal growth factor: determination of the polypeptide backbone chain fold by nuclear magnetic resonance and distance geometry. Proceedings of the National Academy of Sciences of the USA 8-t: 5226-5230. Montclionc GT, Winkler ME, Burton LE. Rinderknecht E, Sporn ME & Wagner G (1989) Sequence-specific lH-NMR assignments and identification of two small antiparallcl J3 sheets in the solution structure of recombinant human transforming growth factor alpha. Proceedings of the National Academy of Sciences of the USA 86: 1519--1523. Moolenaar WH. Yardcn T, de Laat SW & Schlessinger J (1982) Epidermal growth factor induces electrically silent Na" influx in human fibroblasts. Journal of Biological Chemistry 257: 8502-8506. Mroczkowski B, Mosig G & Cohen S (198-1) ATft-stimulatcd interaction between epidermal growth factor receptor and supcrcoilcd DNA. Nature 309: 270--273.

EGF AND TGF-a

567

Mroczkowski B, Reich M, Chen K. Bell GI & Cohen S (1989) Recombinant epidermal growth factor precursor is a glycosylated membrane protein with biological activity.lIIolecularand Cellular Biology 9: 2771-2778. Nexo E, Hollenberg M. Figueroa A & Pratt RM (1980) Detection of EGF-urogastrone and its receptor in fetal mouse development. Proceedings ofthe National Academy ofSciences of the USA 77: 2782-2785. Olsen PS. Nexo E. Poulsen SS. Hansen HF & Kirkegaard P (1984) Renal origin of rat urinary epidermal growth factor. Regulatory Peptides 10: 37-H. Pascali JC, Jones DSC. Doel SM. Clements JM. Hunter M. Fallon T. Edwards M & Brown KD (1991) Cloning and characterization of a gene encoding pig epidermal growth factor. Journal of Molecular Endocrinology 6: 63--70. Pennica D. Holmes WE. Kohr WJ. Harkins RN. Vehar GA. Ward CA. Bennett WF. Yelverton E. Seeburg PH. Heyneker HL. Goeddel DV & Collen D (191)3) Cloning and expression of human tissue-type plasminogen activator cON A in E. coli. Nature 301: 21.f--22 I. Perheentupa J. Lakshmann J & Fisher DA (1984) Epidermal growth factor in neonatal mouse urine: maturative effect of thyroxine. Pediatric Research 18: 1080-101>4. Perheentupa J. Lakshmann J & Fisher DA (1985) Urine and kidney epidermal growth factor: ontogeny and sex difference in the mouse. Pediatric Research 19: 428--B2. Pike U, Marquardt H. Todaro GJ. Gallis B, Casnellie JE. Bornstein P & Krebs EG (1982) Transforming growth factor and epidermal growth factor stimulate the phosphorylation of a synthetic tyrosine-containing peptide in a similar manner. Journal of Biological Chemistry 257: 14628-14631. Porter CD & Archard LC (191)7) Characterization and physical mapping of Molluscum contagiosum virus DNA and location of a sequence capable of encoding a conserved domain of epidermal growth factor. Journal of General Virology 68: 673--61>2. Rakowicz-Szulczyska EM, Otwiaska D, Rodeck U & Koprowski II (191)9) Epidermal growth factor (EGF) and monoclonal antibody to cell surface EGF receptor bind to the same chromatin receptor. Archives of Biochemistry and Biophysics 268: 456-t64. Rail LB, Scott J, Bell GI, Crawford RJ, Penschow JD, Niall HD & Coghlan JP (1985) Mouse prepro-epidermal growth factor synthesis by the kidney and other tissues. Nature 313: 228-231. Reisner AH (1985) Similarity between the vaccinia virus 19k early protein and epidermal growth factor. Nature 313: 801-803. Rozengurt E (1983) Growth factors. cell proliferation and eancer; an overview. Molecular Biology and Medicine I: 169--181. Samsoondar J, Kobrin MS & Kudlow JE (1986) a transforming growth factor secreted by untransformed bovine anterior pituitary cells in culture. Journal of Biological Chemistry 261: 14408-14413. Savage CR & Cohen S (1972) Epidermal growth factor and a new derivative: rapid isolation procedures and biological and chemical characterization. Journal of Biological Chemistry 247: 7609--7611. Savage CR. Inagami T & Cohen S (1972) The primary structure of epidermal growth factor. Journal of Biological Chemistry 247: 7612-7621. Savage CR, Hash JH & Cohen S (1973) Epidermal growth factor: location of disulphide bonds. Journal of Biological Chemistry 248: 7669--7672. Savion N, Vlodavsky I & Gospodarowicz D (1980) Role of the degradation process in the mitogenic effect of epidermal growth factor. Proceedings of the National Academy of Sciences of the USA 77: 1466-1470. Schlessinger J, Shechter Y, Willingham MC & Past an I (1978) Direct visualization of binding, aggregation and internalization of insulin and epidermal growth factor on living fibroblastic cells. Proceedings of the National Academy of Sciences of the USA 75: 2659--2663. Scott J, Urdea M, Quiroga M, Sanchcz-Pescador R, Fong N, Selby M. Rutter WJ & Bell GI (1983) Structure of mouse submaxillary messenger RNA encoding epidermal growth factor and seven related proteins. Science 221: 238-240. Scott J, Patterson S, Rail L, Bell GI, Crawford R, Penschow J, Niall H & Coughlan J (191)5) The structure and biosynthesis of epidermal growth factor. Journal of Cell Science supplement 3: 19--28.

568

c.

A. BROWNE

Server AC & Shooter EM (1976) Comparison of the arginine esteropeptides associated with the nerve and epidermal growth factors. Journal of Biological Chemistry 251: 165-173. Server AC, Sutter A & Shooter EM (1976) Modification of the epidermal growth factor affecting the stability of its high molecular weight complex. Journal of Biological Chemistry 251: 1188-1196. Shechter Y, Schlessinger J, Jacobs S, Chong KJ & Cuatrecasas P (1978a) Fluorescent labelling of hormone receptors in viable cells: preparation and properties of highly fluorescent derivatives of epidermal growth factor and insulin. Proceedings ofthe National Academy of Sciences of the USA 75: 2135-2139. Shechter Y, Hernaez L & Cuatrecasas P (1978b) Epidermal growth factor: biological activity requires persistent occupation of high-affinity cell surface receptors. Proceedings of the National Academy of Sciences of the USA 75: 5788-5791. Shoyab M.. McDonald VL, Bradley JG & Todaro GJ (1988) Amphiregulin: a bifunctional growth-modulating glycoprotein produced by the phorbol12-myristate 13-acetate-trcated human breast adenocarcinoma cell line MCF-7. Proceedings of the National Academy of Sciences of the USA 85: 6528-6532. Shoyab M, Plowman GD, McDonald VL, Bradley JG & Todaro GJ (1989) Structure and function of human amphiregulin: a member of the epidermal growth factor family. Science 243: 1074-1076. Simpson RJ, Smith JA, Moritz RL, O'Hare MJ, Rudnand PS, Morrison JR, Lloyd CJ, Greyo B, Burgess AW, Nice EC & Cobley UT (1985) Rat epidermal growth factor: complete amino acid sequence. Homology with the corresponding murine and human proteins; isolation of a form truncated at both ends with full in vitro biological activity. European Journal of Biochemistry 153: 629-637. Snead ML, Luo W, Oliver P, Nakamura M, WheelerGD, Bessem C, Bell GF, Rail LB, Slavkin HC (1989) Localization of epidermal growth factor present in tooth and lung during embryonic mouse development. Developmental Biology 134: 420-429. Soderquist AM & Carpenter G (1984) Glycosylation of the epidermal growth factor receptor in A-431 cells. The contribution of carbohydrate to receptor function. Journal of Biological Chemistry 259: 12586-12594. Stoscheck CM, Soderquist AM & Carpenter G (1985) Biosynthesis of the epidermal growth factor receptor in cultured human cells. Endocrinology 116: 528-535. Stroobant P, Rice AP, Gullick WJ, Cheng DJ, Kerr 1M & Waterfield MD (1985) Purification and characterization of vaccinia virus growth factor. Cell 42: 383-393. Sudhof TC, Russell DW, Goldstein JL, Brown MS, Sanchez-Pescador R & Bell GI (1985) Cassette of eight exons shared by genes for LDL receptor and EGFprecursor. Science 228: 893-895. Tam JP (1985) Physiological effects of transforming growth factor in the newborn mouse. Science 229: 673-675. Tanaka T, Har-EI R & Tanzer M (1988) Partial structure of the gene for chicken cartilage proteoglycan core protein. Journal of Biological Chemistry 263: 15831-15835. Taylor JM, Cohen S & Mitchell WM (1970) Epidermal growth factor: high and low molecular weight forms. Proceedings of the National Academy of Sciences of the USA 67: 164-171. Taylor JM, Mitchell WM & Cohen S (1972) Epidermal growth factor: physical and chemical properties. Journal of Biological Chemistry 247: 5928-5934. Taylor JM, Mitchell WM & Cohen S (1974) Characterization of the high molecular weight form of epidermal growth factor. Journal of Biological Chemistry 249: 3198-3203. Thorburn GD, Waters MJ, Young JR, Dolling M, Buntine D & Hopkins PS (1981) Epidermal growth factor: a critical factor in fetal maturation. Ciba Foundation Symposium 86: 172-186. Todaro GJ, Lee DC & De Larco JE (1980) Transforming growth factors produced by certain human tumour cells: polypeptides that interact with epidermal growth factor receptors. Proceedings of the National Academy of Sciences of the USA 77: 5258-5262. Twardzik DH (1985) Differential expression of transforming growth factor-a during prenatal development of the mouse. Cancer Research 45: 5413-5416. Ullrich A, Coussens L, Hayflick J, Dull T, Gray A, Tam A, Lee J, Yarden Y, Liverrnann T, Schlessinger J, Downard J, Mayes E, White N, Waterfield M & Seeburg P (1984) Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature 309: 418-425.

EGF AND TGF-a

569

Ushiro H & Cohen S (1980) Identification of phosphotyrosine as a product of epidermal growth factor-activated protein kinase in A-431 cell membranes. Journal ofBiological Chemistry 255: 8363-8365. Wahl M & Carpenter G (1988) Regulation of epidermal growth factor stimulated formation of inositol phosphates in A431 cells by calcium and protein kinase C. Jot/mal of Biological Chemistry 263: 7581-7590. Weber W, Gill GN & Spiess J (1984) Production of an epidermal growth factor receptor-related protein. Science 224: 294-297. Wong ST, Winchell LF, McCune BK, Earp HS, Teixido J, Massague J, Hermon B & Lee DC (1989) The TGFa precursor expressed on the cell surface binds to the EGF receptor on adjacent cells, leading to signal transduction. Cell 56: 495-506. Wood SA & Kaye PL (1989) Effects of epidermal growth factor on preimplantation mouse embryos. Journal of Reproduction and Fertility 85: 575-582. Yamamoto T, Nishida T, Miyajima N, Kawai S, Ooi T & Toyoshima K (1983) The erbB gene of avian erythroblastosis virus is a member of the src gene family. Cell 35: 71-78. Yang SO, Chou CK, Huang S, Song JS & Chen HC (1989) Epidermal growth factor induces activation of protein kinase FA and ATP, Mg-dependent protein phosphatase in A431 cells. Journal of Biological Chemistry 264: 5407-5·H1. Ye Y-K, Lin Y-Z & Tam JP (1988) Shope fibroma virus growth factor exhibits epidermal growth factor activities in newborn mice. Biochemical and Biophysical Research Communications 154: 497-501.