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Origins of Growth Factors: NGF and EGF

      Growing up on the streets of Brooklyn, I remember being interested in how things worked: taking apart an old telephone and the gears of my new 4-speed bicycle was most enjoyable. As a biology major at Brooklyn College in 1945, I was fascinated by embryology. How does an egg turn into a chicken or a frog or a person? My only insight into the problem was the thought that it was necessary to understand the chemical reactions inside the egg and embryo and not simply observe biological structures. I, therefore, became a double major in chemistry and biology (also with the hope of earning a living as a laboratory technician). My first job after graduating was working nights as a bacteriologist in a milk processing plant. One of my professors at Brooklyn College wrote asking whether I was interested in going to graduate school. The college had been sending one student a year to the Biology Department at Oberlin to work as a laboratory assistant while studying for a master's degree. Because Oberlin offered to pay my tuition plus some $300 a semester for living expenses, I jumped at the chance. School and science were both interesting and fun. Upon graduating, I continued my education toward a Ph.D. working as an assistant in the Biochemistry Department of the University of Michigan under Howard B. Lewis. My research involved studying the Krebs urea cycle in the common earthworms that I collected from the university campus. I had been told at Oberlin that the yellow cells surrounding the gut in the worm corresponded to the mammalian liver. I decided to check whether the enzymatic reactions in worms were similar to those in mammals. They were (
      • Cohen S.
      • Lewis H.B.
      The nitrogenous metabolism of the earthworm (Lumbricus terrestris). II. Arginase and urea synthesis.
      ).
      My first “real job” was in the Pediatrics Department at the University of Colorado studying creatine metabolism in premature infants under Harry Gordon. I think he hired me because he was impressed by my ability to stomach tube earthworms. After several years, I decided I needed to learn the then new technique of using radioisotopes in metabolic studies, and I obtained an American Cancer Society fellowship to work in the Radiology Department of Washington University under Martin Kamen where I combined this new knowledge with my interest in embryology. Although fertilized frog eggs and early embryos are impermeable to small molecules (amino acids, phosphates, etc.), sufficient amounts of C14O2 were metabolized by intact eggs and embryos to permit the identification of the radioactive compounds present at various stages of development (
      • Cohen S.
      The metabolism of C14O2 during amphibian development.
      ).
      Upon completion of my fellowship, Dr. Kamen recommended me for a position in the Zoology Department of Washington University in the laboratory of Viktor Hamburger and Rita Levi-Montalcini. This move was critical in determining the direction of my research for the next 40-some odd years: the isolation, structure, and function of the first of the “growth factors,” nerve growth factor (NGF) and epidermal growth factor (EGF).

      Nerve Growth Factor

      The original neuroembryological experiments started more than half a century ago in the laboratories of Drs. Viktor Hamburger and Rita Levi-Montalcini. They had discovered and were interested in understanding the following phenomenon in the chick embryo. If a chick limb bud is extirpated, the nerve cells that would have enervated the limb die. If an extra limb bud is transplanted to another embryo, new nerve fibers will enter the transplanted limb. Their research focused on how the periphery affected nerve fiber growth.
      One of Dr. Hamburger's former students, Elmer Buecker, reported that if he grafted a fragment of a mouse tumor (Sarcoma 180) onto the body wall of a 3-day chick embryo, sensory nerve fibers entered the tumor. He concluded that the sarcoma was a favorable field for growth of sensory fibers. Dr. Levi-Montalcini, upon her arrival in St. Louis from her laboratory in Italy, decided to repeat this experiment. She found that both sympathetic and sensory fibers penetrated the tumor and also reached tissues they were not supposed to enter. Levi-Montalcini performed other experiments; instead of placing the tumor in the embryo, she put it where there were no nerve fibers (on the chorioallantoic membrane) and obtained the same results. She concluded that a soluble, diffusible agent was released from the tumor and stimulated the growth of the nerve cells. She then devised a hanging drop tissue culture system on glass slides in which a sensory ganglion from a chick embryo was placed in a plasma clot near a fragment of tumor. After 1 day of culture, she observed a cluster of nerve fibers emerging from the ganglion directed toward the tumor.
      Neither Dr. Hamburger nor Dr. Levi-Montalcini were biochemists so they went to several of Washington University's departments looking for a biochemist “mad” enough to study this problem. I was interested in embryology and knew biochemistry so I thought, “I'll have a go at it.”
      It was a good combination; I knew very little about neuroembryology, and they knew very little about biochemistry so we interacted but never argued. As a biochemist, I ground up the tumor and made an extract. It worked! A crude extract prepared from the tumor made nerve fibers grow out from chick sensory ganglia when placed in culture (
      • Cohen S.
      • Levi-Montalcini R.
      Purification and properties of a nerve growth-promoting factor isolated from mouse sarcoma 180.
      ). I thought the active agent was a protein because it was heat-labile, non-dialyzable, and inactivated by a protease but not by DNase or RNase.
      During the 6-year period I labored on the problem, I continued my education in biochemistry by participating in the daily seminar and luncheon led by Arthur Kornberg in the Microbiology Department. Discussing papers in a group that included Paul Berg, Mel Cohen, Jerry Hurwitz, David Hogness, Dale Kaiser, Martin Kamen, and Bob Lehman was an education that could hardly have been improved. When I presented my results to this group, it was Kornberg who suggested I was dealing with a virus and that I should add some phosphodiesterase to destroy all known nucleic acids. I obtained some crude phosphodiesterase from one of his faculty members (Osamu Hayaishi) who was purifying it from snake venom. I incubated the enzyme preparation with my tumor extract and tested for nerve growth activity. The next day I saw the most incredible culture I had ever seen. The ganglion in culture had produced a massive number of nerve fibers in a single day. I first thought that the phosphodiesterase had removed an inhibitor that might have been present in the tumor extract. But no! In a control experiment it was the phosphodiesterase preparation itself that induced nerve fiber outgrowth. Further tests showed that it was not the diesterase but an impurity; a second protein was present in the preparation, which induced nerve growth. We purified this new biologically active protein from snake venom (
      • Cohen S.
      Purification and metabolic effects of a nerve growth-promoting protein from snake venom.
      ); it was thousands of times more potent than our best tumor-derived preparation. Its activity in cultures was inhibited by commercially available anti-snake venom serum, and amazingly, when Dr. Levi-Montalcini injected the purified venom protein into the yolk sac of chick embryos, it produced nerve fiber outgrowth identical to that produced by the transplanted tumor (
      • Levi-Montalcini R.
      • Cohen S.
      In vitro and in vivo effects of a nerve growth-stimulating agent isolated from snake venom.
      ). I then pondered, “What could be the connection between nerve growth, tumors, and snake venom?” Nothing obvious—but I thought “where does snake venom come from, a modified salivary gland?” So, at random, I tested extracts of the salivary gland taken from a male mouse and found that it was just as potent as the snake venom protein in inducing nerve growth in cultures.
      Crude extracts of the male mouse salivary gland were injected daily into newborn mice to test whether it affected nerve growth in the animal. Three effects were noted; not only did the sympathetic ganglia become greatly enlarged, but unexpectedly the mice opened their eyes earlier than normal (as early as 7 days after birth rather than the normal 12–14 days) and their teeth erupted earlier. Furthermore, a rabbit antiserum prepared against the purified salivary gland nerve growth factor, when injected into newborn mice, destroyed most of the sympathetic ganglion cells—a phenomenon subsequently called immunosympathectomy. These experiments indicated that the nerve growth factor indeed had a function in the developing animal (
      • Cohen S.
      Purification of a nerve growth-promoting protein from the mouse salivary gland and its neuro-cytotoxic antiserum.
      ,
      • Levi-Montalcini R.
      • Cohen S.
      Effects of the extract of the mouse submaxillary salivary glands on the sympathetic system of mammals.
      ,
      • Cohen S.
      Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal.
      ).

      Epidermal Growth Factor

      At this point in my life I had started a family and decided it was time to get a more stable position rather than be supported on someone's research grant. As an assistant professor in the Biochemistry Department at Vanderbilt, I decided to leave the nerve growth area and study why crude extracts of the male mouse salivary gland induced early eyelid opening in the mouse. (The purified nerve growth factor did not affect eyelid opening.) My thought was that anything that enhanced a normal developmental process had to be of interest.
      First, I examined histological sections of the eyelid area in control and treated animals. It became clear that precocious eyelid opening was because of an enhancement of epidermal growth and keratinization (
      • Cohen S.
      • Elliott G.A.
      The stimulation of epidermal keratinization by a protein isolated from the submaxillary gland of the mouse.
      ). Based on the eyelid-opening assay, we were able to isolate the protein responsible for this effect and to determine the sequence and disulfide linkage of this 53-amino acid polypeptide—now known as epidermal growth factor (EGF) (
      • Cohen S.
      Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal.
      ,
      • Taylor J.M.
      • Mitchell W.M.
      • Cohen S.
      Epidermal growth factor, physical and chemical properties.
      ,
      • Savage Jr., C.R.
      • Inagami T.
      • Cohen S.
      The primary structure of epidermal growth factor.
      ,
      • Savage Jr., C.R.
      • Hash J.H.
      • Cohen S.
      Epidermal growth factor: location of disulfide bonds.
      ). The question of whether EGF acted directly on the epidermis or via a secondary hormonal mechanism in the intact animal was answered by the observation that its direct addition to organ cultures of chick embryonic skin led to a marked increase in epidermal cell number and size (
      • Cohen S.
      The stimulation of epidermal proliferation by a specific protein (EGF).
      )—hence the name.
      Our laboratory then spent a number of years examining some of the metabolic effects of EGF on epidermal cultures (
      • Hoober J.K.
      • Cohen S.
      Epidermal growth factor I. The stimulation of protein and ribonucleic acid synthesis in chick embryo epidermis.
      ,
      • Hoober J.K.
      • Cohen S.
      Epidermal growth factor II. Increased activity of ribosomes from chick embryo epidermis for cell-free protein synthesis.
      ,
      • Cohen S.
      • Stastny M.
      Epidermal growth factor III. The stimulation of polysome formation in chick embryo epidermis.
      ,
      • Stastny M.
      • Cohen S.
      Epidermal growth factor IV. The induction of ornithine decarboxylase.
      ,
      • Sawyer S.T.
      • Cohen S.
      Enhancement of calcium uptake and phosphatidylinositol turnover by epidermal growth factor in A-431 cells.
      ). I also distributed EGF to a number of other laboratories to test in their systems. Pedro Cuatrecasas discovered that, in culture, EGF stimulated the growth of human fibroblasts. I therefore thought there must be a receptor on human cells that could bind mouse EGF and also there must be an EGF-like molecule present in humans.
      By this time we had another method for detecting and measuring EGF—a radio receptor assay using human fibroblasts and 125I-labeled EGF in culture. By this method, we discovered that human urine contained an EGF-like molecule and we were able to purify it. This human protein, derived from urine, made human fibroblasts grow in culture; when we injected it into mice it made their eyelids open earlier. Therefore, we called it human EGF (hEGF). It was of similar size to murine EGF but slightly different in amino acid composition (
      • Carpenter G.
      • Lembach K.J.
      • Morrison M.M.
      • Cohen S.
      Characterization of the binding of 125I-labeled epidermal growth factor to human fibroblasts.
      ,
      • Cohen S.
      • Carpenter G.
      Human epidermal growth factor: isolation and chemical and biological properties.
      ,
      • Carpenter G.
      • Cohen S.
      125I-Labeled human epidermal growth factor: binding, internalization and degradation in human fibroblasts.
      ,
      • Starkey R.A.
      • Cohen S.
      • Orth D.N.
      Epidermal growth factor: identification of a new hormone in human urine.
      ).
      At this same time in England, Dr. Gregory was investigating, as a possible ulcer treatment, a substance in urine (called urogastrone) that when injected into animals inhibited acid secretion in the stomach. From thousands of liters of urine they isolated urogastrone and found that it was a small protein that unexpectedly was identical to our human EGF, both in chemical composition and biological activity. Both urogastrone and hEGF inhibited acid secretion and stimulated cell growth.
      In our laboratory we pursued the question: how does EGF make a cell grow and divide? In those days, it was thought that protein hormones did not enter cells but attached themselves to the receptors on the cell surface and then left after performing their task. We were able to label EGF and, thus, follow its fate after it bound to its receptor on the cell surface. (Three labeling methods were used: attaching radioactive iodine, a fluorescent molecule, or ferritin to the EGF molecule.) We finally concluded that EGF and its receptor were internalized into the cell in small membrane vesicles that eventually were degraded in lysosomes. However, we could not decide whether this internalization served merely to remove the receptor from the cell surface (desensitization) or was part of a signaling mechanism (
      • Haigler H.
      • Ash J.F.
      • Singer S.J.
      • Cohen S.
      Visualization by fluorescence of the binding and internalization of epidermal growth factor in human carcinoma cells A-431.
      ,
      • Gorden P.
      • Carpentier J.L.
      • Cohen S.
      • Orci L.
      Epidermal growth factor: morphological demonstration of binding, internalization, and lysosomal association in human fibroblasts.
      ,
      • Haigler H.T.
      • McKanna J.A.
      • Cohen S.
      Direct visualization of the binding and internalization of a ferritin conjugate of epidermal growth factor in human carcinoma cells A-431.
      ,
      • McKanna J.A.
      • Haigler H.T.
      • Cohen S.
      Hormone receptor topology and dynamics: morphological analysis using ferritin-labeled epidermal growth factor.
      ,
      • Cohen S.
      • Fava R.A.
      Internalization of functional epidermal growth factor: receptor/kinase complexes in A-431 cells.
      ).
      As a biochemist I thought a hormone or growth factor should regulate some chemical reaction in a cell that might be demonstrable in a cell-free system. The only successful experiment in those days was Earl Sutherland's demonstration that adding glucagon to cell membranes stimulated the synthesis of cyclic AMP.
      Fortunately, I had a friend at the National Institutes of Health (Dr. Joe DeLarco) who was surveying all of their stored cells for EGF receptors. He discovered and told me about a human squamous carcinoma cell (A431) that had millions of receptors per cell rather than the normal number of thousands per cell. I thought it should be easier to detect, in a cell-free system, a reaction that was regulated by EGF and its receptor. Membranes prepared from A431 cells were capable of binding massive amounts of EGF. The presence of EGF altered the morphology of these cells (
      • Chinkers M.
      • McKanna J.A.
      • Cohen S.
      Rapid induction of morphological changes in human carcinoma cells A-431 by epidermal growth factor.
      ) and stimulated pinocytosis (
      • Haigler H.T.
      • McKanna J.A.
      • Cohen S.
      Rapid stimulation of pinocytosis in human carcinoma cells A-431 by epidermal growth factor.
      ). When we incubated these membranes with 32P-labeled ATP, radioactivity was incorporated into the protein of the membrane. The addition of EGF to the incubation mixture greatly stimulated this reaction (
      • Carpenter G.
      • King Jr., L.
      • Cohen S.
      Rapid enhancement of protein phosphorylation in A-431 cell membrane preparations by epidermal growth factor.
      ).
      As a biochemist, I was exhilarated! A cell-free system where EGF has an effect. When we examined what was phosphorylated in the membranes (by SDS-gel electrophoresis and autoradiography), we saw that in the presence of EGF, many proteins were phosphorylated, especially a protein of 170 kDa (
      • King Jr., L.E.
      • Carpenter G.
      • Cohen S.
      Characterization by electrophoresis of epidermal growth factor stimulated phosphorylation using A-431 membranes.
      ). A protein of this size had been suggested by Das and Fox to be the molecular weight of the receptor for EGF. To isolate the receptor for EGF, I made an affinity column to which EGF was linked. Membranes from A-431 cells were solubilized in detergent and passed through the column. Only the solubilized receptor remained “stuck” on the column. It could be eluted by competition with soluble EGF and isolated. The isolated 170-kDa protein was able to bind EGF, had protein kinase activity dependent on ATP and EGF, and was itself phosphorylated. We concluded that the receptor was a transmembrane protein with at least three domains: an external EGF-binding domain, an internal kinase domain, and a domain that acted as a substrate which could be phosphorylated (
      • Cohen S.
      • Carpenter G.
      • King Jr., L.
      Epidermal growth factor-receptor-protein kinase interactions: co-purification of receptor and epidermal growth factor-enhanced phosphorylation activity.
      ,
      • Cohen S.
      • Ushiro H.
      • Stoscheck C.
      • Chinkers M.A.
      A native 170,000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles.
      ,
      • Buhrow S.A.
      • Cohen S.
      • Staros J.V.
      Affinity labeling of the protein kinase associated with the epidermal growth factor receptor in membrane vesicles from A-431 cells.
      ,
      • Erneux C.
      • Cohen S.
      • Garbers D.L.
      The kinetics of tyrosine phosphorylation by the purified epidermal growth factor receptor kinase of A-431 cells.
      ,
      • Buhrow S.A.
      • Cohen S.
      • Garbers D.L.
      • Staros J.V.
      Characterization of the interaction of 5′-p-fluorosulfonylbenzoyl adenosine with the epidermal growth factor receptor/protein kinase in A-431 cell membranes.
      ,
      • Russo M.W.
      • Lukas T.J.
      • Cohen S.
      • Staros J.V.
      Identification of residues in the nucleotide binding site of the epidermal growth factor/receptor/kinase.
      ).
      Originally we thought that threonine was the amino acid phosphorylated, because our analytical method did not distinguish it from tyrosine. When Tony Hunter reported a new method that could separate phosphothreonine from phosphotyrosine and that the transforming protein of the Rous sarcoma virus was a tyrosine kinase and not a threonine kinase, we reinvestigated the EGF receptor and determined that it, too, was a tyrosine kinase (
      • Ushiro H.
      • Cohen S.
      Identification of phosphotyrosine as a product of epidermal growth factor-activated protein kinase in A-431 cells.
      ). Originally it had been thought that the transforming protein was a foreign enzyme that interacted with the tyrosine residue and that was what caused cancer. However, the EGF receptor is a normal cellular protein with a normal cellular function. Nevertheless, the relationship was supported by the finding that antibodies to the Rous sarcoma transforming protein interacted with the EGF receptor (
      • Chinkers M.
      • Cohen S.
      Purified EGF receptor-kinase interacts specifically with antibodies to Rous sarcoma virus transforming protein.
      ).
      Our laboratory continued to explore the metabolic responses to EGF both in cell culture (
      • Sawyer S.T.
      • Cohen S.
      Enhancement of calcium uptake and phosphatidylinositol turnover by epidermal growth factor in A-431 cells.
      ,
      • Sawyer S.J.
      • Cohen S.
      Epidermal growth factor stimulates the phosphorylation of the calcium-dependent 35,000-dalton substrate in intact A-431 cells.
      ,
      • De B.K.
      • Misono K.S.
      • Lukas T.J.
      • Mroczkowski B.
      • Cohen S.
      A calcium-dependent 35-kilodalton substrate for epidermal growth factor receptor/kinase isolated from normal tissue.
      ,
      • Giugni T.D.
      • Chen K.
      • Cohen S.
      Activation of a cytosolic serine protein kinase by epidermal growth factor.
      ,
      • McKanna J.A.
      • Cohen S.
      The EGF receptor kinase substrate p35 in the floor plate of the embryonic rat CNS.
      ,
      • Donaldson R.W.
      • Cohen S.
      Epidermal growth factor or okadaic acid stimulates phosphorylation of eukaryotic initiation factor 4F.
      ) and in intact animals, aided by the discovery that phosphotyrosyl protein phosphatase activity was inhibited by vanadate (
      • Swarup G.
      • Cohen S.
      • Garbers D.L.
      Inhibition of membrane phosphotyrosyl-protein phosphatase activity by vanadate.
      ,
      • Ruff S.J.
      • Chen K.
      • Cohen S.
      Peroxovanadate induces tyrosine phosphorylation of multiple signaling proteins in mouse liver and kidney.
      ). A number of EGF-dependent intracellular phosphorylated substrates were detected including the annexins (
      • Fava R.A.
      • Cohen S.
      Isolation of a calcium-dependent 35-kilodalton substrate for the epidermal growth factor receptor/kinase from A-431 cells.
      ,
      • Furge L.L.
      • Chen K.
      • Cohen S.
      Annexin VII and Annexin XI are tyrosine-phosphorylated in peroxovanadate-treated dogs and in platelet-derived growth factor-treated rat vascular smooth muscle cells.
      ), STAT proteins (
      • Donaldson R.W.
      • Cohen S.
      Epidermal growth factor stimulates tyrosine phosphorylation in the neonatal mouse: association of Mr 55,000 substrate with the receptor.
      ,
      • Ruff-Jamison S.
      • Zhong Z.
      • Wen Z.
      • Chen K.
      • Darnell Jr., J.E.
      • Cohen S.
      Epidermal growth factor and lipopolysaccharide activate Stat3 transcription factor in mouse liver.
      ,
      • Ruff-Jamison S.
      • Chen K.
      • Cohen S.
      Epidermal growth factor induces the tyrosine phosphorylation and nuclear translocation of Stat5 in mouse liver.
      ,
      • Quelle F.W.
      • Thierfelder W.
      • Witthuhn B.A.
      • Tang B.
      • Cohen S.
      Phosphorylation and activation of the DNA binding activity of purified Stat1 by the Janus protein-tyrosine kinases and the epidermal growth factor receptor.
      ), and SHC (
      • Ruff-Jamison S.
      • McGlade J.
      • Pawson T.
      • Chen K.
      • Cohen S.
      Epidermal growth factor stimulates the tyrosine phosphorylation of SHC in the mouse.
      ,
      • Donaldson R.W.
      • Cohen S.
      Epidermal growth factor stimulates tyrosine phosphorylation in the neonatal mouse: association of Mr 55,000 substrate with the receptor.
      ). With regard to prepro-EGF, its presence in mouse kidney membranes and human urine as well as its biological activity were confirmed by isolation (
      • Breyer J.A.
      • Cohen S.
      The epidermal growth factor precursor isolated from murine kidney membranes.
      ,
      • Parries G.
      • Chen K.
      • Misono K.S.
      • Cohen S.
      The human urinary epidermal growth factor (EGF) precursor.
      ); its cDNA was expressed as a glycosylated membrane protein in NIH 3T3 cells (
      • Mroczkowski B.
      • Reich M.
      • Chen K.
      • Bell G.I.
      • Cohen S.
      Recombinant human epidermal growth factor precursor is a glycosylated membrane protein with biological activity.
      ,
      • Mroczkowski B.
      • Reich M.
      • Whittaker J.
      • Bell G.I.
      • Cohen S.
      Expression of human epidermal growth factor precursor cDNA in transfected mouse NIH 3T3 cells.
      ).

      Epilogue

      Since the initial discoveries of NGF and EGF, thousands of related papers and numerous reviews have been published revealing many new aspects of growth regulation. I can only mention a few. The receptors of many other growth factors and hormones have been found to be specific transmembrane, ligand-activated tyrosine kinases including the receptors for insulin and NGF; EGF-related loci are found in insects and round worms; the amino acid sequence of the transforming protein of the erythroblastosis virus is closely related to the amino acid sequence of the kinase domain of the EGF receptor; a family of EGF-related molecules has been isolated; at least four EGF-related receptors, given various names, have been detected and one of them (Erb2) is overexpressed in some human breast tumors. This has led to more targeted clinical therapy.
      None of the findings described in this brief personal history could have been possible without the efforts of many colleagues, students, and technicians, to all of whom I am most grateful. In the reference list of the publications from this laboratory, I have provided the titles of each paper to indicate the research area to which each investigator contributed. I also wish to acknowledge with gratitude the support of the National Institutes of Health and the American Cancer Society. Finally, on a personal note, I am both amazed and encouraged by the unpredictable scientific and clinical consequences of simply wondering what caused precocious eyelid openings in newborn mice.

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        Purification and metabolic effects of a nerve growth-promoting protein from snake venom.
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        Purification of a nerve growth-promoting protein from the mouse salivary gland and its neuro-cytotoxic antiserum.
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        Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal.
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        Epidermal growth factor: location of disulfide bonds.
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        Characterization of the binding of 125I-labeled epidermal growth factor to human fibroblasts.
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        Human epidermal growth factor: isolation and chemical and biological properties.
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        Visualization by fluorescence of the binding and internalization of epidermal growth factor in human carcinoma cells A-431.
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