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A collection of cell lines expressing each human epidermal growth factor receptor (HER) family member alone or in all pairwise combinations in a clone of NIH3T3 cells (3T3-7d) devoid of detectable epidermal growth factor receptor family members has been generated. Transformation, as measured by growth in soft agar, occurred only in the presence of appropriate ligand and only in cells expressing two different HER family members. Transfection of oncogenic neu (Tneu), conferred ligand-independent transformation only in cells which co-expressed HER1, HER3, or HER4, but not when expressed alone or with HER2. Cell lines were also tested for their ability to form tumors in animals. None of the cell lines expressing single HER family members was able to form tumors in animals with the exception of HER1, which was weakly tumorigenic. Although unable to form tumors when expressed alone, HER2 was tumorigenic when expressed with HER1 or HER3, but not HER4. Of all complexes analyzed, cells expressing HER1 + HER2 were the most aggressive. The relationship between HER1 activation, intracellular calcium fluxes, and phospholipase Cgamma1 activation is well established. We found that activation of HER1 was required for the induction of a calcium flux and the phosphorylation of phospholipase Cgamma1. These activities were independent of, and unaffected by, the co-expression of any other family member. Further, heregulin stimulation of all cell lines including those containing HER1 did not demonstrate any effect on intracellular calcium levels or phospholipase Cgamma1 phosphorylation. This demonstrates that heregulin induced cellular transformation by activating HER3- and HER4-containing complexes does not require the activation of either phospholipase Cgamma1 or the mobilization of intracellular calcium.

We recently reported the molecular cloning of HER4/p180erbB4, a new member of the epidermal growth factor receptor family, as well as its activation by a partially purified ligand (Plowman, G. D., Culouscou, J.-M., Whitney, G. S., Green, J. M., Carlton, G. W., Foy, L., Neubauer, M. G., and Shoyab, M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1746-1750). In this report we describe the purification to homogeneity of a 45-kDa protein (p45) that induces the differentiation of MDA-MB-453 human breast cancer cells and stimulates the tyrosine phosphorylation of p180erbB4, the HER4-encoded protein. Hydrophobic interaction, ion-exchange, heparin, and size exclusion chromatographies were used to purify this p180erbB4 activator to homogeneity. N-terminal amino acid sequencing suggests that p45 is related to heregulin, a recently reported ligand for p185erbB2. Binding and cross-linking experiments demonstrated that p45 specifically binds to cells expressing recombinant p180erbB4 and not cells expressing recombinant p185erbB2.

This report describes the isolation and recombinant expression of a cDNA clone encoding HER4, the fourth member of the human epidermal growth factor receptor (EGFR) family. The HER4/erbB4 gene encodes a 180-kDa transmembrane tyrosine kinase (HER4/p180erbB4) whose extracellular domain is most similar to the orphan receptor HER3/p160erbB3, whereas its cytoplasmic kinase domain exhibits 79% and 77% identity with EGFR and HER2/p185erbB2, respectively. HER4 is most predominantly expressed in several breast carcinoma cell lines, and in normal skeletal muscle, heart, pituitary, brain, and cerebellum. In addition, we describe the partial purification of a heparin-binding HER4-stimulatory factor from HepG2 cells. This protein was found to specifically stimulate the intrinsic tyrosine kinase activity of HER4/p180erbB4 while having no direct effect on the phosphorylation of EGFR, HER2, or HER3. Furthermore, this heparin-binding protein induces phenotypic differentiation, and tyrosine phosphorylation, of a human mammary tumor cell line that overexpresses both HER4 and HER2. These findings suggest that this ligand-receptor interaction may play a role in the growth and differentiation of some normal and transformed cells.

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.

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.

Insecticyanin was found to be synthesized in several isoelectric forms and stored in the pigment granules in the epidermis. Both major epidermal forms (INS-a, pl 5.5; INS-b, pl 5.7) were found in the cuticle, but only the most basic form, INS-b, was present in the hemolymph. In vitro the epidermis synthesized and secreted both forms into both the cuticle and the medium. Isolation of two cDNA clones for insecticyanin followed by hybridization to epidermal mRNA showed the presence of only one 1.1 kb mRNA, but transcription of the longer cDNA yielded a RNA which produced INS-a but no INS-b. Insecticyanin mRNA was present during the intermolt feeding stages of the 4th and 5th instars and absent during the larval molt and after the onset of metamorphosis. Exposure of either day 2 4th-instar or day 1 5th-instar larval epidermis to 20-hydroxyecdysone (20HE) in vitro caused a dose-dependent decline in this mRNA that was not prevented by simultaneous exposure to JH. When synthesis resumes just before ecdysis, INS-b appears before INS-a; then on the final day of feeding, synthesis of INS-a ceases before that of INS-b. Culture experiments showed that exposure of day 15th epidermis to a small pulse of 20HE followed by an ecdysteroid-free period was best to mimic the in vivo situation, indicating that the small rise of ecdysteroid in the absence of JH on day-2 was responsible for this differential cessation.

We have constructed, expressed, and purified a fusion protein, HAR-TX beta 2, consisting of heregulin-beta 2 fused to a binding-defective form of Pseudomonas exotoxin A, PE40. The fusion protein was found to induce receptor tyrosine phosphorylation in CEM cells transfected with HER4 alone or in combination with HER2 but not in cells transfected with HER2 or HER1 alone. The phosphorylation of receptor tyrosines was both dose-dependent and saturable in amounts similar to those shown to be active for native heregulin. HAR-TX beta 2 was specifically cytotoxic toward a variety of carcinoma cell lines in the ng/ml range. However, some tumor cell lines were found to be insensitive to the cytotoxic action of the fusion protein even at > 2 micrograms/ml. Relative amounts of HER4, HER3, and HER2 were determined on seven cell lines sensitive and four cell lines insensitive to HAR-TX beta 2. All lines that express HER4 were killed by HAR-TX beta 2, while none lacking HER4 were affected. HAR-TX beta 2 was able to bind to and signal via tyrosine phosphorylation in cell lines that co-express HER2 and HER3 in the absence of HER4 without inducing cytotoxicity. Thus HAR-TX beta 2 may prove to be a useful reagent for the targeting and elimination of HER4-positive tumor cells.

The HER4/ERBB4 gene encodes a 180K transmembrane protein (HER4/p180erbB4) that is structurally related to the 185K product (HER2/p185erbB2) of the HER2/ERBB2 proto-oncogene. A 45K heparin-binding glycoprotein (p45) has been characterized that specifically activates the intrinsic tyrosine kinase activity of HER4 (ref. 2). This HER4 ligand shares several features with the heregulin family of proteins, including molecular mass, ability to induce differentiation of breast cancer cells, activation of tyrosine phosphorylation in MDA-MB453 cells, and amino-terminal protein sequence. Heregulin exists as multiple isoforms and all are presumed to interact directly with HER2 (refs 3-6). We have used binding and phosphorylation studies with recombinant ligand on cell lines expressing recombinant receptors, and report here that heregulin, like p45, is a specific ligand for HER4. Furthermore, heregulin fails to induce phosphorylation of HER2 in the absence of HER4. These findings suggest that activation of the HER4 receptor is involved in signal transduction by heregulin.

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.

The absence of juvenile hormone at the time of head cap slippage during the last-larval moult of the tabacco hornworm, Manduca sexta, causes deposition of premelanin granules into the outer regions of the newly forming endocuticle beginning 13 h later. These granules were found to contain an inactive phenoloxidase which becomes activated about 9 h later, 4 h before body melanization begins. The onset of melanization was not accelerated by melanization and reddish colouration hormone from Bombyx heads, extracts of pharate-adult corpora cardiaca or pharate-larval ventral nerve cords (sources of eclosion hormone), or extracts of pharate-larval suboesophageal ganglia or corpora cardiaca-corpora allata complexes. Instead the fall of the ecdysteroid titre to below 250 ng/ml 20-hydroxyecdysone equivalents appeared to be the cue that allowed melanization about 4.5 h later. Up to, but not after, this time both melanization and ecdysis could be delayed by exogenous 20-hydroxyecdysone in a dose-dependent fashion above 0.1 μg per larva. In vitro studies published elsewhere indicate that 20-hydroxyecdysone prevents the activation of the premelanin granules. Thus the granules can be deposited at the proper time in the newly forming endocuticle but their melanization is regulated by the declining ecdysteroid titre and it thus synchronized with other events occurring just before ecdysis.

Of a series of derivatives of JH I and methoprene, iodovinylmethoprenol (IVMA) proved most active ( ed 50 s of 3.2 pmol and 20 nM in the Manduca black larval assay and in the prevention of the 20-hydroxyecdysone-induced change to pupal commitment in the epidermis in vitro , respectively). When incubated with nuclei isolated from day 1 fifth instar abdominal epidermis, [ 125 I]IVMA bound specifically to two components with K d s of 4 and 59 nM. This binding was competed by IVMA and methoprene but not by JH I; similar binding by [ 3 H]JH I was not competed by IVMA or methoprene. Specific binding of IVMA was not found in pupally committed epidermis from wandering larvae, but it reappeared in pupal abdominal and thoracic epidermis but not in the wings. Culture of day 2 fifth instar epidermis with 20-hydroxyecdysone to cause pupal commitment caused the loss of the IVMA nuclear binders whereas culture in the absence of hormortes had no effect, indicating that 20HE in the absence of JH downregulates JH receptors.

When epidermis from wandering stage tobacco hornworm ( Manduca sexta ) larvae was exposed to 5 μg/ml 20-hydroxyecdysone for 3 days, then exposed to hormone-free Grace's medium, the newly formed pupal cuticle tanned slowly up to 35% of its area by day 12. The addition of 1.3 mM dopamine on either day 4 or 5 slightly increased the area tanned and addition of β-alanine (to 11.2 mM) on days 3–5 enhanced tanning 2–2.5-fold by day 8. Later addition had no effect. When pharate pupal cuticle about 24 h before ecdysis was explanted to Grace's medium, little tanning occurred in 24 h unless dopa or dopamine or their derivatives were added; β-alanine up to 4.4 mM had no effect. Partial tanning occurred in 10 mM dopa or dopamine. More effective were N -β-alanylnorepinephrine and N -β-alanyldopamine which produced nearly maximal tanning at 1 and 5 mM respectively. Up to 10 mM N -β-acetylnorepinephrine had little effect. Thus, dopamine and β-alanine are important to cuticular tanning in vitro and apparently need to be incorporated into the exocuticle during its synthesis. Maximal tanning of this exocuticle then requires further incorporation of β-alanyl conjugates.