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1.
Science ; 243(4894 Pt 1): 1074-6, 1989 Feb 24.
Artículo en Inglés | MEDLINE | ID: mdl-2466334

RESUMEN

The complete amino acid sequence of amphiregulin, a bifunctional cell growth modulator, was determined. The truncated form contains 78 amino acids, whereas a larger form of amphiregulin contains six additional amino acids at the amino-terminal end. The amino-terminal half of amphiregulin is extremely hydrophilic and contains unusually high numbers of lysine, arginine, and asparagine residues. The carboxyl-terminal half of amphiregulin (residues 46 to 84) exhibits striking homology to the epidermal growth factor (EGF) family of proteins. Amphiregulin binds to the EGF receptor but not as well as EGF does. Amphiregulin fully supplants the requirement for EGF or transforming growth factor-alpha in murine keratinocyte growth, but it is a much weaker growth stimulator in other cell systems.


Asunto(s)
Glicoproteínas/fisiología , Sustancias de Crecimiento/fisiología , Péptidos y Proteínas de Señalización Intercelular , Secuencia de Aminoácidos , Anfirregulina , Animales , Unión Competitiva , División Celular , Familia de Proteínas EGF , Células Epidérmicas , Factor de Crecimiento Epidérmico/fisiología , Receptores ErbB/metabolismo , Humanos , Queratinas/metabolismo , Ratones , Datos de Secuencia Molecular , Ensayo de Unión Radioligante , Homología de Secuencia de Ácido Nucleico , Factores de Crecimiento Transformadores/fisiología
2.
Mol Cell Biol ; 10(5): 1969-81, 1990 May.
Artículo en Inglés | MEDLINE | ID: mdl-2325643

RESUMEN

We have isolated the gene for a novel growth regulator, amphiregulin (AR), that is evolutionarily related to epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha). AR is a bifunctional growth modulator: it interacts with the EGF/TGF-alpha receptor to promote the growth of normal epithelial cells and inhibits the growth of certain aggressive carcinoma cell lines. The 84-amino-acid mature protein is embedded within a 252-amino-acid transmembrane precursor, an organization similar to that of the TGF-alpha precursor. Human placenta and ovaries were found to express significant amounts of the 1.4-kilobase AR transcript, implicating AR in the regulation of normal cell growth. In addition, the AR gene was localized to chromosomal region 4q13-4q21, a common breakpoint for acute lymphoblastic leukemia.


Asunto(s)
Glicoproteínas/genética , Sustancias de Crecimiento/genética , Péptidos y Proteínas de Señalización Intercelular , Secuencia de Aminoácidos , Anfirregulina , Secuencia de Bases , Northern Blotting , Cromosomas Humanos Par 4 , Clonación Molecular , ADN/genética , Familia de Proteínas EGF , Factor de Crecimiento Epidérmico/genética , Genes , Humanos , Datos de Secuencia Molecular , Conformación Proteica , Precursores de Proteínas/genética , ARN Mensajero/genética , Mapeo Restrictivo , Distribución Tisular , Factores de Crecimiento Transformadores/genética
3.
Growth Factors ; 7(4): 305-14, 1992.
Artículo en Inglés | MEDLINE | ID: mdl-1306686

RESUMEN

Amphiregulin (AR) is a 23 kDa, bifunctional growth modulating glycoprotein belonging to the epidermal growth factor (EGF) family of polypeptide growth regulators. AR possesses two putative nuclear localization sequences (NLS), binds to DNA sepharose, and localizes to the nucleoli of human ovarian surface epithelial carcinoma cells suggesting that AR has a direct nuclear role. We have found that 125I-labeled AR, when exogenously applied to several carcinoma cell lines, associated with nuclei in a time, temperature, and concentration dependent fashion. The control peptide, EGF, also associated with these fractions but at approximately 20% of the efficiency of AR. Cross-linking experiments with 125I-labeled AR and nuclear fractions derived from various carcinoma and normal cell lines demonstrated that AR binds two proteins of molecular mass 205 and 120 kDa. AR binding to these nuclear fraction proteins was specific and saturable as shown by competition experiments utilizing both SV-40 large T antigen NLS and an AR derived peptide encompassing both putative AR NLS. The combined results suggest that nuclear interactions may play a significant role in AR induced growth responses.


Asunto(s)
Núcleo Celular/metabolismo , Glicoproteínas/metabolismo , Sustancias de Crecimiento/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Proteínas Nucleares/metabolismo , Secuencia de Aminoácidos , Anfirregulina , Animales , Unión Competitiva , Transporte Biológico , Fraccionamiento Celular , Nucléolo Celular/metabolismo , Familia de Proteínas EGF , Femenino , Humanos , Cinética , Datos de Secuencia Molecular , Señales de Localización Nuclear , Ratas , Temperatura , Células Tumorales Cultivadas
4.
Biochem Biophys Res Commun ; 179(1): 572-8, 1991 Aug 30.
Artículo en Inglés | MEDLINE | ID: mdl-1883381

RESUMEN

Amphiregulin-associated protein (ARAP) was purified from serum-free conditioned medium of MCF-7, human breast carcinoma cells, treated with 12-0-tetradecanoylphorbol-13-acetate (TPA). ARAP is a single-chain, extremely hydrophilic, heparin-binding protein. Its apparent molecular weight is approximately 21,500 as assessed by gel chromatography and approximately 15,500 as determined by polyacrylamide gel electrophoresis. The complete amino acid sequence of ARAP was determined. The larger form contains 123 amino acids, whereas a shorter form is missing two amino acids at the amino-terminal. ARAP contains 10 cysteines and 30 basic amino acids (23 lysines and 7 arginines). ARP sequence has been found to be identical to protein encoded by human MK gene.


Asunto(s)
Proteínas Portadoras/biosíntesis , Factores de Crecimiento Nervioso , Biosíntesis de Proteínas , Acetato de Tetradecanoilforbol/farmacología , Adenocarcinoma , Secuencia de Aminoácidos , Animales , Neoplasias de la Mama , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Línea Celular , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Midkina , Datos de Secuencia Molecular , Peso Molecular , Conformación Proteica , Proteínas/genética , Proteínas/aislamiento & purificación , Homología de Secuencia de Ácido Nucleico
5.
Proc Natl Acad Sci U S A ; 87(20): 7912-6, 1990 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-2236009

RESUMEN

Two proteins, termed epithelin 1 and epithelin 2, that inhibit the growth of A431 cells, derived from a human epidermal carcinoma of the vulva, have been purified from rat kidney. Epithelin 1 stimulates the proliferation of murine keratinocytes, whereas epithelin 2 inhibits the epithelin 1-elicited growth of these cells. Thus epithelin 1 and 2 behave as agonist and antagonist, respectively, for normal epithelial cells. Epithelins are low molecular mass (approximately 6 kDa), acid- and heat-stable, single-chain proteins containing approximately 20% cysteine. Some of these cysteines form disulfide linkage(s) that are essential for biological activity. The amino-terminal amino acid sequences of epithelin 1 and epithelin 2 have been determined. The two proteins showed no substantial sequence homology with other proteins. However, a significant homology was seen between the amino-terminal sequences of epithelin 1 and epithelin 2. Epithelins 1 and 2, therefore, appear to represent members of a distinct family of growth regulators.


Asunto(s)
Cisteína , Replicación del ADN/efectos de los fármacos , Inhibidores de Crecimiento/aislamiento & purificación , Sustancias de Crecimiento/aislamiento & purificación , Péptidos y Proteínas de Señalización Intercelular , Queratinocitos/citología , Riñón/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Granulinas , Inhibidores de Crecimiento/farmacología , Sustancias de Crecimiento/farmacología , Humanos , Queratinocitos/efectos de los fármacos , Riñón/química , Cinética , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Ratas
6.
Proc Natl Acad Sci U S A ; 85(17): 6528-32, 1988 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-3413110

RESUMEN

A glycoprotein, termed amphiregulin (AR), inhibits growth of several human carcinoma cells in culture and stimulates proliferation of human fibroblasts and certain other tumor cells. It has been purified to apparent homogeneity from serum-free conditioned medium of MCF-7 human breast carcinoma cells that had been treated with phorbol 12-myristate 13-acetate. AR is a single-chain extremely hydrophilic glycoprotein containing cysteines in disulfide linkage(s) that are essential for biological activity; it is stable between pH 2 and pH 12 and after heating for 30 min at 56 degrees C but unstable at 100 degrees C. The apparent molecular weights of AR and N-Glycanase-treated AR are 14,000 and 15,000, respectively, as assessed by gel chromatography, and approximately 22,500 and approximately 14,000, respectively, as determined by polyacrylamide gel electrophoresis. Treatment of AR with N-Glycanase, O-Glycanase, or neuraminidase does not affect its activity. The pI of AR is approximately 7.8. The amino-terminal amino acid sequence of AR has been determined, and no significant sequence homology between AR and other proteins was found. The molecule thus appears to be a distinct growth regulatory protein.


Asunto(s)
Adenocarcinoma/metabolismo , Neoplasias de la Mama/metabolismo , Glicoproteínas/biosíntesis , Sustancias de Crecimiento/biosíntesis , Péptidos y Proteínas de Señalización Intercelular , Acetato de Tetradecanoilforbol/farmacología , Secuencia de Aminoácidos , Anfirregulina , Animales , División Celular/efectos de los fármacos , Línea Celular , Replicación del ADN/efectos de los fármacos , Familia de Proteínas EGF , Glicoproteínas/aislamiento & purificación , Glicoproteínas/farmacología , Sustancias de Crecimiento/aislamiento & purificación , Sustancias de Crecimiento/farmacología , Humanos , Datos de Secuencia Molecular
7.
Growth Factors ; 9(3): 167-75, 1993.
Artículo en Inglés | MEDLINE | ID: mdl-8274294

RESUMEN

Human melanoma cell line A375 is extremely sensitive to growth inhibitory effects of oncostatin M (OM). A375 cells resistant to the antiproliferative effect of OM were isolated by exposing OM-sensitive cells to ethyl methane sulfonate (EMS) for 24 h followed by continuous exposure to OM. An A375 subline resistant to OM-induced growth inhibition was selected by a limiting dilution technique and designated 4-1.10". The resistant cells were completely refractory to OM even up to a concentration of 500 ng/ml. Interestingly, the resistant cells were also nonresponsive to the growth inhibitory effects of interleukin-6 (IL-6). Other cytokines such as transforming growth factor beta (TGF-beta), interleukin-1 (IL-1), and tumor necrosis factor alpha and beta (TNF-alpha and TNF-beta) exhibited similar growth inhibitory effects on OM-sensitive or -resistant cells. OM-resistant cells were found to possess approximately 20% of OM receptors with the same affinities as compared to the parental OM-sensitive cells. However, the affinities and number of receptors for IL-6 were the same on both cell types. The OM treatment did not alter the cyclic AMP (cAMP) level of either the parental or the resistant cells. The OM-resistant cell line will be very useful in elucidating the mechanism of OM-elicited growth inhibition.


Asunto(s)
Inhibidores de Crecimiento/farmacología , Interleucina-6/farmacología , Péptidos/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Antineoplásicos/farmacología , División Celular/efectos de los fármacos , AMP Cíclico/fisiología , Humanos , Melanoma , Oncostatina M , Receptores de Interleucina/metabolismo , Receptores de Interleucina-6 , Transducción de Señal
8.
Proc Natl Acad Sci U S A ; 87(13): 4905-9, 1990 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-2164210

RESUMEN

Epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), and amphiregulin are structurally and functionally related growth regulatory proteins. These secreted polypeptides all bind to the 170-kDa cell-surface EGF receptor, activating its intrinsic kinase activity. However, amphiregulin exhibits different activities than EGF and TGF-alpha in a number of biological assays. Amphiregulin only partially competes with EGF for binding EGF receptor, and amphiregulin does not induce anchorage-independent growth of normal rat kidney cells (NRK) in the presence of TGF-beta. Amphiregulin also appears to abrogate the stimulatory effect of TGF-alpha on the growth of several aggressive epithelial carcinomas that overexpress EGF receptor. These findings suggest that amphiregulin may interact with a separate receptor in certain cell types. Here we report the cloning of another member of the human EGF receptor (HER) family of receptor tyrosine kinases, which we have named "HER3/ERRB3." The cDNA was isolated from a human carcinoma cell line, and its 6-kilobase transcript was identified in various human tissues. We have generated peptide-specific antisera that recognizes the 160-kDa HER3 protein when transiently expressed in COS cells. These reagents will allow us to determine whether HER3 binds amphiregulin or other growth regulatory proteins and what role HER3 protein plays in the regulation of cell growth.


Asunto(s)
Receptores ErbB/genética , Expresión Génica , Genes , Proteínas Tirosina Quinasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular/métodos , Humanos , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Conformación Proteica , Homología de Secuencia de Ácido Nucleico , Transfección , Células Tumorales Cultivadas/enzimología
9.
J Biol Chem ; 267(18): 13073-8, 1992 Jun 25.
Artículo en Inglés | MEDLINE | ID: mdl-1618805

RESUMEN

Epithelin 1 and 2 were originally purified from rat kidneys based on their ability to inhibit the growth of A-431 human epidermoid carcinoma cells (Shoyab, M., McDonald, V.L., Byles, C., Todaro, G.J., and Plowman, G.D. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7912-7916). This study presents the complete amino acid sequence of these two growth factors and the cloning of their cDNA from rat, mouse, and human sources. Epithelins 1 and 2 are 56- and 57-amino acid polypeptides, respectively, and share 47% amino acid sequence identity with the conserved spacing of 12 cysteine residues. Molecular cloning revealed that both proteins are encoded by a single precursor that contains 7 1/2 copies of this novel 12-cysteine motif, 2 of which represent the known active molecules. Recombinant expression in COS cells demonstrated that the epithelin 1 protein was mitogenic on rodent keratinocytes and fibroblasts. In contrast, epithelin 2 had no activity on these cells, but at high concentrations was capable of antagonizing the growth proliferative activities of epithelin 1. Northern analysis shows the epithelin mRNA to be expressed in many types of epithelial cells. The broad expression profile of epithelin transcripts, along with the opposing activities of the two mature protein products, implicates these factors as natural mediators of epithelial homeostasis.


Asunto(s)
División Celular , Endotelinas/genética , Péptidos y Proteínas de Señalización Intercelular , Precursores de Proteínas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Línea Celular , Clonación Molecular , ADN , Endotelina-1 , Endotelinas/metabolismo , Células Epiteliales , Expresión Génica , Granulinas , Inhibidores de Crecimiento/genética , Inhibidores de Crecimiento/metabolismo , Sustancias de Crecimiento/genética , Sustancias de Crecimiento/metabolismo , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Precursores de Proteínas/metabolismo , Ratas , Alineación de Secuencia
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