Epidermal Growth Factor *

Growth factors, a diverse group of polypeptides that modify cell proliferation, constitute a distinct subgroup in endocrinology. The biology of these factors differs somewhat from classical hormones as neither their site(s) of synthesis nor site(s) of action is restricted to defined tissues. Many growth factors probably operate in a paracrine fashion and, in certain instances, their action may be autocrine in nature. The diffuseness of sites of synthesis and location of target cells plus the limited quantities of purified material available for studies with intact animals have restricted progress in understanding the normal physiological function of many of the growth factors in growth and development. However, early studies with EGF’ in the intact animal demonstrated its stimulatory effect on epidermal proliferation (1) and inhibitory effect on gastric acid secretion (2). The application of biochemical and molecular biological approaches has produced considerable information concerning the structure of the growth factors and their individual receptors, their classification into families of related molecules, the relationship of receptors and growth factors to oncogene products, and the plethora of cellular events that constitute the mitogenic response. Also, some clues are available regarding the second messenger pathways that mediate biological responses to growth factors. The study of EGF has provided a framework for understanding the cellular and molecular events that underlie the biological effects of a number of growth factors and hormones. The intent of this article is to summarize this information with reference to the most seminal discoveries and recent advances. A recent comprehensive review (3) is available for more detailed information.

Growth factors, a diverse group of polypeptides that modify cell proliferation, constitute a distinct subgroup in endocrinology. The biology of these factors differs somewhat from classical hormones as neither their site(s) of synthesis nor site(s) of action is restricted to defined tissues. Many growth factors probably operate in a paracrine fashion and, in certain instances, their action may be autocrine in nature. The diffuseness of sites of synthesis and location of target cells plus the limited quantities of purified material available for studies with intact animals have restricted progress in understanding the normal physiological function of many of the growth factors in growth and development.
However, early studies with EGF' in the intact animal demonstrated its stimulatory effect on epidermal proliferation (1) and inhibitory effect on gastric acid secretion (2).
The application of biochemical and molecular biological approaches has produced considerable information concerning the structure of the growth factors and their individual receptors, their classification into families of related molecules, the relationship of receptors and growth factors to oncogene products, and the plethora of cellular events that constitute the mitogenic response. Also, some clues are available regarding the second messenger pathways that mediate biological responses to growth factors.
The study of EGF has provided a framework for understanding the cellular and molecular events that underlie the biological effects of a number of growth factors and hormones. The intent of this article is to summarize this information with reference to the most seminal discoveries and recent advances. A recent comprehensive review (3) is available for more detailed information.

Structure of EGF and Its Relatives
While the primary and secondary structures of EGF have been known for some time (4, 5), three advances in this area have been made in the last few years. First is the realization that there is a family of EGF-like molecules, all of which are encoded by distinct genes. The other members of this family are TGFcu, the pox virus growth factors, and amphiregulin (3). These EGF-like molecules are defined by three characteristics: high affinity binding to the EGF receptor, production of mitogenic responses in EGF-sensitive cells, and within the primary structure of approximately 50-60 residues, 6 halfcystines in the general sequence X,CX&X2_3GXC X10.13CXCXZYXGXRCX,LX,.
Interestingly, the motif of half-cystine residues in this mitogen family is also found in a  (3), do have interesting properties in development, cell adhesion, and protein-protein interactions. A second advance has come from NMR studies of EGF (6)(7)(8) and TGFa (9)(10)(11). The results reveal that both polypeptides have two sets of anti-parallel P-sheet structures, but little or no cr-helical conformation.
While attempts have been made to synthesize biologically active peptides corresponding to various portions of the EGF molecule, significant successes have not been reported.
However, it has been possible to synthesize the entire EGF (12) and TGFa (13) molecules with full biologic activity. The results of site-directed mutagenesis indicate that all half-cystine residues are essential for biologic activity (3).
The third advance in this area relates not to the structure of the mature EGF molecule but to the surprising structure of its precursor.
cDNA cloning revealed that prepro-EGF contains approximately 1200 residues (14,15). The sequence of this precursor includes not only the sequence of EGF but also eight EGF-like units and, near the carboxyl terminus, a hydrophobic sequence characteristic of an integral membrane protein. Subsequent studies with transfected cells have demonstrated that prepro-EGF can exist as a glycosylated membrane protein (16). The means by which EGF is processed from the precursor molecule is not known, and there is substantial interest in the other functions of the precursor. Whereas in the mouse submaxillary gland the EGF precursor is rapidly processed to the 53-amino acid form of EGF, in certain cells of the kidney the precursor accumulates and does not appear to be processed intracellularly to mature EGF (17). The kidney is postulated to be the source of urinary EGF. Interestingly, the intact EGF precursor, purified either from mouse kidney* or cultured cells transfected with cDNA for prepro-EGF (16), retains EGF-like biological activity.

Structure and Function of the EGF Receptor
As shown in Fig. 1, the mature EGF receptor, M, = 170,000, is composed of a single polypeptide chain of 1186 amino acid residues and a substantial amount (approximately 40,000 daltons) of N-linked oligosaccharide.
A single hydrophobic membrane anchor sequence separates an extracellular ligandbinding domain from a cytoplasmic domain that encodes an EGF-regulated tyrosine kinase (18,24,25). cDNA cloning of the chicken EGF receptor has revealed approximately 80% identity to the human EGF receptor sequence (31). The basic organizational motif of the EGF receptor is not unlike that of receptors for several other growth factors (PDGF, insulin, insulin-like growth factor 1, colony stimulating factor 1, and fibroblast growth factor). The cytoplasmic tyrosine kinase domain of these growth factor receptors is similar to a substantial number of oncogene products. Tyrosine kinase activity, therefore, has a central role in the regulation of cell proliferation.
The extracellular domain of the EGF receptor is characterized by its capacity to bind EGF and EGF-like ligands with high affinity. Chemically this portion of the receptor contains lo-11 N-linked oligosaccharide chains (26,27), an unusually indicate cysteine residues, and the stippled area denotes w-like tyrosine kinase sequences (18). P-Y designates tyrosine autophosphorylation sites (19, ZO), P-T and P-S designate phos II hothreonine and phosphorserine residues (21,22), respectively, and A'Z'P-K enotas a lysine residue critical for ATP binding (23).
high content of half-cystine residues (10%) that could give rise to as many as 25 disulfides, and, in several cell lines, mannose phosphate (28). It is proposed that the region between the two half-cystine-rich clusters is involved in ligand binding (29). The hallmark of the cytoplasmic portion of this receptor is the sequence defining the tyrosine kinase domain. This domain has particularly high homology to the avian erb B oncogene products (18) which are, in fact, derived from the avian gene for the EGF receptor (30). Near the carboxyl terminus of the receptor are four sites of EGF-dependent tyrosine autophosphorylation (19,20). Present data suggest that these COOH-terminal tyrosines define an autoinhibitory region that can be relieved by autophosphorylation or truncation (32, 33).
Treatment of intact cells with EGF produces a marked increase in the formation of phosphoserine, phosphothreonine, and phosphotyrosine on the EGF receptor. The nontyrosine phosphorylations are attributable to nonreceptor kinases such as protein kinase C, suggesting that these kinases are indirectly stimulated by ligand binding to the receptor and, in a possible feedback loop, utilize the receptor as one of their substrates. Seven non-tyrosine phosphorylation sites have been identified bordering the tyrosine kinase domain (21,22). Of these serine/threonine phosphorylation sites it is clear that C kinase phosphorylates threonine 654 (21) and probably one or more other sites that are not yet identified. C kinase phosphorylation of the receptor produces attenuating effects on the tyrosine kinase domain and, in some cells, on the ligand-binding domain (3).
A major point regarding the structure and function of the EGF receptor is the molecular mechanism by which ligand binding activates the tyrosine kinase domain. Evidence has been presented for an EGF-induced oligomerization mechanism (34) coupled with intermolecular phosphorylation (35). The molecular details of this hypothesis remain unclear and alternate concepts, ie. the mechanism is entirely intramolecular, have been advanced.

Signal Transduction
Lysine 721 of the EGF receptor participates in ATP binding and is essential for enzyme activity (23). Mutagenesis of this residue has demonstrated that, although ligand binding properties are not altered, all measurable cellular responses to EGF are abrogated (36, 37). Therefore, tyrosine kinase activity following ligand binding is essential and the first step in the EGF signal transduction pathway.
Recently, substantial progress has been made in identifying tyrosine kinase substrates that have known biochemical functions. This permits construction of a potential mitogenic signaling pathway (Fig. 2). This map depicts five proteins as tyrosine kinase substrates (PLC--yl, PI-3 kinase, GAP, MAP kinase, and raf kinase). Two others, lipocortin I (calpactin, pp35) (39-41) and c-erb B-2 (38,(42)(43)(44), are not represented. The latter is an EGF receptor-like molecule that possesses 1 EFF ] 63 H / \?, - Epidermal Growth Factor 7711 tyrosine kinase activity and an extracellular binding site for an as yet unidentified growth factor. Lipocortin associates with membrane phospholipids in the presence of calcium; however, its physiological function is unknown. The best characterized substrate of the EGF receptor is PLC-rl, one of a family of isozymes that hydrolyzes PI 4,5bisphosphate to produce IPs and diacylglycerol (45). The former acts as a second messenger molecule to liberate stored calcium from the endoplasmic reticulum and thereby activates calcium-requiring enzymes or processes, while the latter is an activator of protein kinase C. Within 1 min after the addition of EGF to cells, approximately 60% of the total PLC--rl molecules are phosphorylated on tyrosine residues (46-48). This growth factor-enhanced phosphorylation of PLC-71 occurs mostly at tyrosine sites, but some increase in serine phosphate, presumably catalyzed by an EGF-activated serine kinase, is also produced. In uitro, the purified EGF receptor phosphorylates PLC-yl on tyrosine residues but produces little or no phosphorylation of phospholipases @ or 6 (49). In contrast, the insulin receptor, which also possesses tyrosine kinase activity, does not utilize PLC-71 as a substrate (48, 50). This is a striking level of specificity for tyrosine phosphorylation in vitro, suggestive of biologic significance (55).
PLC-~1 is a unique PLC isozyme in that it contains sequences shared with several cytoplasmic tyrosine kinases, GAP, and the crk gene product. These sequences are referred to as src homology regions, SH2 and SH3, and may have regulatory functions in this diverse group of growth-related molecules (51). Although sites of tyrosine phosphorylation of PLC-71 have been identified (52,53), none of these phosphotyrosine residues lie precisely within SH regions or the major regions of sequence conservation in the PLC isozyme family.
A second tyrosine kinase substrate involved in phosphoinositide metabolism is PI-3 kinase. Though most phosphoinositides do not contain a phosphate at the 3-position of the inositol ring, a small pool of phosphoinositides bearing this phosphorylation has been identified recently in growth factorstimulated cells (54). These uniquely phosphorylated phosphoinositides are not hydrolyzed by any known phospholipase, and it has been proposed that these phospholipids may serve an alternate function, perhaps as cofactors for membrane-bound enzymes. Tyrosine kinase activation of the PI 3-kinase has been demonstrated for several growth factors. Increased levels of phosphoinositol 3,4-bisphosphate have been reported in one EGF-treated cell line (56). There is, however, no clear demonstration that the enzymatic activity of this PI-3 kinase is regulated by tyrosine phosphorylation.
GAP, an activating protein for the GTPase activity of ras, is another tyrosine kinase substrate that functions at the plasma membrane. Tyrosine phosphorylation of GAP in intact cells is stimulated by several tyrosine kinases, including the EGF receptor (57,58). Though the stoichiometry of GAP tyrosine phosphorylation seems low (less than lo%), tyrosine phosphorylation of GAP coincides with GAP translocation to the membrane. Presumably, membrane-localized GAP is complexed with rus. Two serine kinases have been reported to be tyrosine kinase substrates. MAP kinase is subject to enhanced tyrosine phosphorylation in EGF-treated cells (59). An interesting substrate of MAP kinase is the S6 kinase, which phosphorylates ribosomal protein S6. Phosphorylation of S6 kinase by MAP kinase increases the catalytic activity of the S6 kinase (60). Both MAP and S6 kinases are serine/threonine kinases, and their activities are increased in 61). Whether this cascade actually alters ribosomal function has not been demonstrated, however.
There is substantial interest in the serine kinase rufi the proto-oncogene of the transforming gene of murine sarcoma virus 3611. This molecule is a tyrosine phosphorylation substrate of the PDGF receptor (62), and data show that growth factor-stimulated phosphorylation(s) increases the catalytic activity of ruf (63). Since PDGF increases the level of phosphotyrosine and phosphoserine on raf, it is not possible to ascribe the activation of enzymatic activity to a particular type of phosphorylation. In EGF-treated cells increased phosphorylation of raf has been documented (62), though identification of the phosphoamino acids was not reported. Physiological substrates for raf are not known. The notion that signal transduction involves a cascade of protein kinases is not a new one. Of these kinases, casein kinase II is of particular interest. While the mechanism of EGF activation of casein kinase II (64-66) is not known, this kinase has two properties of interest to nuclear events accompanying growth stimulation. Localization studies show that casein kinase II is found in both the nucleus and cytoplasm (82), and phosphorylation studies (in vitro) indicate that casein kinase II phosphorylates an interesting spectrum of nuclear proteins including enzymes that modify DNA topology (73, 74) and several transcription factors: myc (68), E7 (78), large T antigen (69), SRF (70,71), and c-erb A (72). Phosphorylation in vitro of DNA topoisomerase II by casein kinase II increases topoisomerase activity 3-fold (73), and phosphorylation of SRF enhances DNA binding activity (70, 71).
The scheme presented in Fig. 2 is only a working model. Critically, the transducing elements that activate the transcription of early genes (fos, jun, etc.) are unclear. All of the reactions shown in Fig. 3 occur very rapidly (less than 60 min), but EGF must remain in the extracellular environment for nearly 8 h before increased DNA synthesis becomes committed (67). Also, one needs to recognize that EGF, like other growth factors, elicits biologic responses unrelated to mitogenesis (3) and signaling for these responses may involve new pathways or some subset of the pathways depicted above.
Transfection of the EGF receptor gene into cells that do not otherwise express EGF receptors or respond to EGF has produced interesting results. Addition of EGF to these transfected cells produces a mitogenic response (75). This indicates that other than EGF and the EGF receptor all the necessary signaling components for an EGF response are present. These results would be consistent with a signal transduction pathway utilizing the tyrosine kinase substrates described above.
It has been known for some time that the formation of EGF-receptor complexes on the cell surface is followed by rapid internalization and degradation of ligand (76) and receptor (77). How this pathway relates to signal transduction remains unclear. While it is possible to recover internalized receptors that remain activated in terms of tyrosine kinase activity (79-81), there are also data indicating that mitogenic signaling is enhanced if internalization is slowed (33,83,84). A short segment of the EGF receptor residues 973-991 has been identified as responsible for mediating internalization of EGF-receptor complexes (83).
Prospectus EGF, its receptor, and the general scheme for signal transduction are representative of a large number of growth factors. Variations clearly exist in the structure of growth factors and their receptors, but most all depend on tyrosine kinase activity as the initial step in their mechanism of action. Of course, a major point of interest will be those differences in signaling that exist between growth factor receptors regulating normal