We previously described a novel alternatively spliced mRNA transcript of the betacellulin (BTC) gene. This splice isoform, termed BTC-delta 4, lacks the C-loop of the epidermal growth factor motif and the transmembrane domain as a result of exon 4 'skipping'. In this study, we expressed BTC-delta 4 recombinantly to explore its biological function. When BTC-delta 4 was expressed in COS-7 cells, it was secreted largely into the culture medium, in contrast to BTC. Unlike BTC, highly purified recombinant BTC-delta 4 produced in Escherichia coli failed to bind or induce tyrosine phosphorylation of either ErbB1 or ErbB4, nor did it antagonize the binding of BTC to these receptors. Consistent with this, BTC-delta 4 failed to stimulate DNA synthesis in Balb/c 3T3 and INS-1 cells. However, BTC-delta 4 induced differentiation of pancreatic beta-cells; BTC-delta 4 converted AR42J cells to insulin-producing cells. When recombinant BTC-delta 4 was administered to streptozotocin-treated neonatal rats, it reduced the plasma glucose concentration and improved glucose tolerance. Importantly, BTC-delta 4 significantly increased the insulin content, the beta-cell mass, and the numbers of islet-like cell clusters and PDX-1-positive ductal cells. Thus, BTC-delta 4 is a secreted protein that stimulates differentiation of beta-cells in vitro and in vivo in an apparent ErbB1- and ErbB4- independent manner. The mechanism by which BTC-delta 4 exerts this action on beta-cells remains to be defined but presumably involves an, as yet, unidentified unique receptor.

Betacellulin belongs to the family of epidermal growth factor-like growth factors that are expressed as transmembrane precursors and undergo proteolytic ectodomain shedding to release a soluble mature growth factor. In this study, we investigated the ectodomain shedding of the betacellulin precursor (pro-BTC) in conditionally immortalized wild-type (WT) and ADAM-deficient cell lines. Sequential ectodomain cleavage of the predominant cell-surface 40-kDa form of pro-BTC generated a major (26-28 kDa) and two minor ( 20 and 15 kDa) soluble forms and a cellular remnant lacking the ectodomain (12 kDa). Pro-BTC shedding was activated by calcium ionophore (A23187) and by the metalloprotease activator p-aminophenylmercuric acetate ( APMA), but not by phorbol esters. Culturing cells in calcium-free medium or with the protein kinase Cdelta inhibitor rottlerin, but not with broad-based protein kinase C inhibitors, blocked A23187-activated pro-BTC shedding. These same treatments were without effect for constitutive and APMA-induced cleavage events. All pro-BTC shedding was blocked by treatment with a broad-spectrum metalloprotease inhibitor (GM6001). In addition, constitutive and activated pro-BTC shedding was differentially blocked by TIMP-1 or TIMP-3, but was insensitive to treatment with TIMP-2. Pro-BTC shedding was functional in cells from ADAM17- and ADAM9-deficient mice and in cells overexpressing WT or catalytically inactive ADAM17. In contrast, overexpression of WT ADAM10 enhanced constitutive and activated shedding of pro-BTC, whereas overexpression of catalytically inactive ADAM10 reduced shedding. These results demonstrate, for the first time, activated pro-BTC shedding in response to extracellular calcium influx and APMA and provide evidence that ADAM10 mediates constitutive and activated pro-BTC shedding.

Betacellulin is a relatively new member of the epidermal growth factor peptide family, however, its function remains poorly defined. We investigated its physiological effects in rats implanted with pumps to deliver vehicle or recombinant rat betacellulin [46 7 g/day] for 7 days. At kill, blood and gastrointestinal tissues were collected for determinations of betacellulin levels, proliferation (bromodeoxyuridine-BrdU incorporation) and growth. Plasma betacellulin levels were increased 8-fold compared to vehicle, whilst serum insulin, body weight and food intake were decreased by 32, 15 and 9%, respectively. Water intake, urine and faecal output and small intestinal weight were respectively increased by 36, 78, 47 and 24%. Ileal and proximal colonic crypt depths were increased by 25 and 51% although the BrdU labelling index was unaffected. Betacellulin stimulated gastrointestinal growth, the increased responsiveness of the terminal ileum and colon suggesting therapeutic potential in disease conditions in which ileal or colonic re-growth is desirable. Betacellulin further stimulated a diuresis suggesting an additional role in fluid homeostasis.

Betacellulin (BTC) belongs to the epidermal growth factor (EGF) family of peptide ligands that are characterized by a six-cysteine consensus motif (EGF-motif) that forms three intra-molecular disulfide bonds, crucial for binding the ErbB receptor family. A variety of in vitro studies have identified BTC as an important factor in the growth and/or differentiation of pancreatic islet cells. The molecular mechanisms that regulate the transcription of the BTC gene however have not been delineated. As an initial step, we have characterized the genomic structure of the mouse BTC (mBTC) gene. mBTC cDNA was used as a probe to screen a mouse 129/SVJ genomic bacterial artificial chromosome (BAC) library. Three positive clones containing the entire gene were isolated. DNA sequence analysis identified six exons (1–6) and five introns (A–E); a structure conserved among the EGF family. PCR analysis showed that introns A–E are approximately 7.8, 8.9, 3.8, 1.4 and 1.4 kb in length, respectively. The EGF-motif is encoded by exons 3 and 4 with an intron (intron C) disrupting the coding sequence between the second and third disulfide loops. All exon–intron boundaries are consistent with the “gt-ag” rule. Multiple transcription start sites and one poly(A) site, located 18 bp downstream of a polyadenylation signal sequence, were identified by 5′- and 3′-RACE, respectively. Approximately 2.6 kb of 5′-flanking region was sequenced and was shown to lack consensus TATA and CCAAT boxes, but was found to contain several putative cis-acting regulatory elements. These included consensus binding sites for transcription factors HNF3β, USF, Nkx2–5, AP-4, and Sp1. Functional promoter analysis of the 5′-flanking region in COS-7 cells, using 5′-deletion fragments (−168/+335; −635/+335; −732/+335; −1175/+335; −1698/+335) cloned into a promoterless firefly luciferase reporter vector, identified basal promoter activity and both positive and negative cis-acting elements.

Betacellulin, a member of the epidermal growth factor (EGF) family, was originally isolated and identified from the conditioned medium from a murine pancreatic beta-cell carcinoma cell line. Recently, we isolated bovine betacellulin from a growth factor enriched cheese whey extract, but there is no information on the presence of betacellulin in other biological fluids. We have cloned the cDNA for bovine betacellulin, produced recombinant betacellulin and shown that it has a similar potency to the purified native molecule in stimulating the proliferation of Balb/c3T3 fibroblasts. We have produced a polyclonal antiserum to bovine betacellulin which did not cross-react with EGF or transforming growth factor-alpha (TGF-alpha). The antibody was used in a homologous RIA that was able to detect betacellulin in pooled bovine colostrum sampled during the first 3 days after calving (2.30+/-0.11 ng/ml mean+/-s.e.m.; n=6), in bovine milk soluble fraction (1.93+/-0.64 ng/ml mean+/-s.e.m.; n=5) and in bovine cheese whey (2.59+/-0.16 ng/ml mean+/-s.e.m.; n=3). The betacellulin concentration in foetal bovine serum (FBS) (3.68+/-0.59 ng/ml mean+/-s.e.m.; n=6) greatly exceeded that of betacellulin in serum from male calves 1 and 5 weeks of age (0.53+/-0.15 ng/ml and 0.70+/- 0.09 ng/ml respectively; mean+/-s.e.m.; n=9). Betacellulin measured in the serum of these same animals when aged between 27 and 43 weeks was below the detection limits of the RIA. Sera from 10 out of 36 unmated heifers contained betacellulin levels within the detection limits of the assay (0.433+/-0.06 ng/ml mean+/-s.e.m.; n=10). The presence of betacellulin in bovine colostrum and milk suggests that it plays a role in the growth and development of the neonate and/or mammary gland function. The results also show that betacellulin is undetectable in the castrated adult male circulation. Additionally, although present in very low amounts, serum betacellulin could be under hormonal regulation in the female, since betacellulin was detected in sera from 27% of the unmated heifers examined in this study. The high levels of betacellulin detected in FBS relative to newborn and adult serum suggests a possible endocrine role for this growth factor in the bovine foetus.

A method for the large scale expression and purification of rat betacellulin (BTC) from Escherichia coli has been developed using a cleavable fusion protein strategy. Insoluble fusion protein collected as inclusion bodies was dissolved in urea under reducing conditions, re-folded, and purified by gel filtration chromatography and C(4) RP-HPLC. Authentic rat BTC was obtained after proteolytic cleavage of the fusion protein with Factor Xa. Factor Xa cleaved an additional site within the BTC protein, generating a truncated isoform separable from full-length BTC by heparin-affinity chromatography. Recombinant rat BTC stimulated the proliferation of mouse Balb/c 3T3 fibroblasts and competed for binding to the ErbB1 receptor in a dose-dependent manner analogous to that of BTC purified from natural sources.

Although arachidonic acid has been demonstrated to stimulate a wide variety of cellular functions, the responsible mechanisms remain poorly defined. We now report that arachidonic acid stimulated the activity of class Ia phosphatidylinositol 3-kinase (PI3K) in human umbilical vein endothelial cells, HL60 cells, and human neutrophils. Pretreatment of endothelial cells with AG-1478, an inhibitor of the ErbB receptor family, resulted in the suppression of PI3K activation by arachidonic acid. The fatty acid enhanced the tyrosine phosphorylation of ErbB4 but not of ErbB2 or ErbB3. The ability of arachidonic acid to stimulate PI3K activity in neutrophils was suppressed by indomethacin and nordihydroguaiaretic acid, inhibitors of the cyclooxygenases and lipoxygenases, respectively, but not by 17-octadecynoic acid, an inhibitor of omega-hydroxylation of arachidonic acid by cytochrome P450 monooxygenases. Consistent with this, the activity of PI3K in neutrophils was stimulated by 5-hydroxyeicosatetraenoic acid. Arachidonic acid also transiently stimulated the phosphorylation of Akt on Thr-308 and Ser-473. Although PI3K was not required for the activation of the mitogen-activated protein kinases, ERK1, ERK2, and p38, in arachidonic acid-stimulated neutrophils, the fatty acid acted via PI3K to stimulate the respiratory burst. These results not only define a novel mechanism through which some of the actions of arachidonic acid are mediated but also demonstrate that, in addition to ErbB1 (epidermal growth factor receptor), ErbB4 can also be transactivated by a non-epidermal growth factor-like ligand.

Betacellulin (BTC) belongs to the epidermal growth factor (EGF) family of peptide ligands that are characterised by a six-cysteine consensus motif that forms three intra-molecular disulfide bonds crucial for binding the ErbB receptor family. BTC was initially described, purified and cloned from a mouse insulinoma cell line. BTC is proteolytically processed from a larger membrane-anchored precursor and is a potent mitogen for a wide variety of cell types. BTC binds and activates ErbB-1 and ErbB-4 homodimers and is further characterised by its unique ability to activate all possible heterodimeric ErbB receptors. BTC is widely expressed in most tissues and various body fluids, including milk. Expression is particularly high in the pancreas where it is thought to play a role in the differentiation of pancreatic beta cells. While much is known about the ErbB receptor binding characteristics of BTC and its effect on a variety of cultured cells under different conditions, the challenge that lies ahead is to determine the role of BTC in vivo. This review will focus on the structure of BTC and the various biological effects ascribed to this member of the EGF family.

Abstract This paper describes the cloning and characterization of a novel cDNA encoding a short form of betacellulin (BTC-β), and reports the expression of this mRNA in a variety of human tissues and cell types. BTC-β is likely the result of alternative splicing. This splicing event leads to the generation of an mRNA encoding an unusual BTC precursor in which the C-loop of the EGF domain and the transmembrane domain are deleted while the remainder of the mature molecule is fused in-frame to the C-terminal cytoplasmic tail.

Abstract Betacellulin (BTC) is relatively a more recently discovered member of the EGF family of growth factors. As a prelude to its expression and functional studies in rat models of gut damagelrepair, we have cloned rat BTC and examined its expression in the gastrointestinal tract. Rat BTC was found to be nearly identical to mouse betacellulin. A single 3 kb mRNA species was detected by Northern blotting, and ribonuclease protection analysis showed that its expression was ubiquitous but low in abundance throughout the gut. BTC mRNA and protein were found expressed in the gastric surface and upper pit epithelium as well as in some cells of gastric glands. In the jejunum, BTC mRNA and protein were localised to the crypt epithelium and in villous goblet cells. In the colon, BTC mRNA and protein were found produced in crypt and surface epithelium as well as in goblet cells. Taken together, the wide spread expression in the gut epithelium and in mucous cells in particular suggests an important and unique role for BTC in the gastrointestinal tract.

The sori of Dictyostelium discoideum (strains SG1, SG2, NC4 and V12) contained more than 100 mM ammonium phosphate. Glutamine synthetase (GS), which could remove ammonia from the sorus, was not present in 2-d-old dormant spores but enzyme activity returned to vegetative levels after spore germination. Based on mRNA blotting, the activity of this enzyme in germinating spores appeared to be transcriptionally controlled. At the same time that GS activity was increasing, ammonia was released from germinating spores. Exogenous ammonium ions at a concentration of 28 mM did not block germination nor modulate GS activity in nascent amoebae. It was concluded that the transcription and translation of GS is not environmentally regulated but is an integral part of the germination process, preparing nascent amoebae for vegetative growth. An exogenous concentration of 69 mM ammonium phosphate could maintain dormancy in spores of strains SG1 and SG2 for at least a week in the absence of any other inhibitory component from the sori. The inhibition was reversible at any time either by dilution or by washing the spores free of the ammonium ion. Spores of strain acg- were not inhibited by 100 mM ammonium phosphate. A model is presented in which GS in prespore cells serves as a sink for ammonia to allow the osmotically sensitive adenylyl cyclase aggregation protein (ACA) to activate protein kinase A (PKA) to induce fruiting-body formation. After fruiting-body formation is complete, the decline in GS and ACA activities in developing spores is offset by their replacement with the osmotically and ammonia-stimulated adenylyl cyclase osmosensor for germination (ACG). Ammonia and discadenine may act as separate signals to synergistically activate PKA by stimulating ACG activity while inhibiting cAMP phosphodiestrase activity in fully dormant spores.

Betacellulin (BTC), a member of the epidermal growth factor (EGF) family of peptide growth factors, was purified from a growth-factor-enriched whey fraction of bovine milk by a combination of ion-exchange chromatography, gel-filtration chromatography, affinity chromatography and reverse-phase HPLC. Bovine BTC (bBTC) had an apparent molecular mass of 21-22 kDa on SDS/PAGE and exists in a glycosylated form. The cDNA encoding bBTC was obtained by a combination of 5' and 3' rapid amplification of cDNA ends ('RACE'). The primary translation product consists of 178 amino acid residues containing a putative signal sequence, a transmembrane domain, the mature BTC domain and a cytoplasmic domain containing a highly hydrophilic Arg-Lys-rich region similar to that of mouse BTC and human BTC. The amino acid sequence of the bBTC precursor was 88% identical with human BTC and 79% identical with mouse BTC. The bBTC gene was found to be expressed in a wide range of tissues, including the mammary gland. The identification of BTC in milk raises the possibility that it has a major role in the growth and development of the neonatal gastrointestinal tract.

The mammalian cation-independent mannose 6-phosphate receptor (CI-MPR) binds mannose 6-phosphate-bearing glycoproteins and insulin-like growth factor (IGF)-II. However, the CI-MPR from the opossum has been reported to bind bovine IGF-II with low affinity (Dahms, N. M., Brzycki-Wessell, M. A., Ramanujam, K. S., and Seetharam, B. (1993) Endocrinology 133, 440-446). This may reflect the use of a heterologous ligand, or it may represent the intrinsic binding affinity of this receptor. To examine the binding of IGF-II to a marsupial CI-MPR in a homologous system, we have previously purified kangaroo IGF-II (Yandell, C. A., Francis, G. L., Wheldrake, J. F., and Upton, Z. (1998) J. Endocrinol. 156, 195-204), and we now report the purification and characterization of the CI-MPR from kangaroo liver. The interaction of the kangaroo CI-MPR with IGF-II has been examined by ligand blotting, radioreceptor assay, and real-time biomolecular interaction analysis. Using both a heterologous and homologous approach, we have demonstrated that the kangaroo CI-MPR has a lower binding affinity for IGF-II than its eutherian (placental mammal) counterparts. Furthermore, real-time biomolecular interaction analysis revealed that the kangaroo CI-MPR has a higher affinity for kangaroo IGF-II than for human IGF-II. The cDNA sequence of the kangaroo CI-MPR indicates that there is considerable divergence in the area corresponding to the IGF-II binding site of the eutherian receptor. Thus, the acquisition of a high-affinity binding site for regulating IGF-II appears to be a recent event specific to the eutherian lineage.

Glutamine synthetase (GS) from the cellular slime mould Dictyostelium discoideum was purified to apparent electrophoretic homogeneity with a final yield of 21.7%. The native enzyme appeared to be a GS-II type enzyme. SDS-PAGE of the final preparation revealed a single band of 43.5 kDa. The enzyme has a native molecular mass of 376 kDa, determined using Superose 6, indicating that the enzyme is likely to be an octamer of identical subunits. Dictyostelium discoideum GS has an optimal temperature of 42.5 degrees C, although it is thermolabile in the absence of L-glutamate and (or) Mg2+-ATP. The enzyme exhibits a K-m for L-glutamate, ATP, and NH4Cl of 2.18, 0.18, and 0.11 mM, respectively, in the L-glutamine synthetic reaction with an optimal pH of 7.9. GS from D. discoideum does not appear to be significantly inhibited by various end products of L-glutamine metabolism, although it is potently inhibited by methionine sulphoximine. These properties are those expected for an enzyme for which the primary function is the assimilation of ammonia.

To examine further the role of the enzyme glutamine synthetase in Dictyostelium discoideum we report here the effects of a specific glutamine synthetase inhibitor, methionine sulfoximine, on the growth and differentiation of this organism. Vegetative AX3 cells grown in the presence of methionine sulfoximine did not complete culmination in the normal time but were blocked at the finger stage. In these cells glutamine synthetase activity was almost completely abolished. However, methionine sulfoximine did not affect the level of glutamine synthetase mRNA, suggesting that there is no link between glutamine synthetase activity and mRNA transcription. Eventually glutamine synthetase activity reappeared and at the time culmination occurred. These results suggest that glutamine synthetase plays an important role in the assimilation of ammonia during the later stages of development in D. discoideum and that this assimilation is necessary for the completion of culmination.

The enzyme glutamine synthetase (GS) of Dictyostelium discoideum is developmentally regulated, preferentially localized in prespore cells and is likely to play an important role in controlling the levels of ammonia, a known morphogen, in this organism. To further investigate the regulation of GS, a portion of the GS gene was isolated and used as a probe to examine the changes in GS mRNA throughout development and the level of GS mRNA in the two precursor cell types. The amino acid sequence of the cloned DNA fragment isolated is highly homologous to other eukaryotic GS genes. DNA blot analysis demonstrated that the GS gene exists as a single copy in D. discoideum . Analysis of RNA indicated that there is a single 1.7 kb GS transcript that increased during development to peak at the initial stages of culmination. Furthermore, GS mRNA was highly localized in prespore cells, which is consistent with a proposed source–sink model for ammonia assimilation in this organism.

The enzyme glutamine synthetase (GS) is described for the first time in Dictyostelium discoideum. The appearance of this enzyme is developmentally regulated. The level of activity is low in vegetative cells and increases more than threefold during differentiation. Furthermore this enzyme is shown to be differentially localized in prespore cells, the specific activity being approximately fourfold higher than in prestalk cells. The enzyme has a pH optimum of 7.8 and 8.2 in the gamma-glutamyltransferase and gamma-glutamylsynthetase assays, respectively, and a temperature optimum of 45 degrees C. Kinetic studies of GS revealed apparent Km values of 5.9 mM, 0.009 mM and 8.6 mM for glutamine, ADP and NH2OH, respectively, in the gamma-glutamyltransferase assay, and of 2.2 mM, 0.12 mM and 0.64 mM for glutamate, ATP and NH2OH, respectively, in the gamma-glutamylsynthetase assay.