Andrew J. Dunbar

Control of ErbB signaling through metalloprotease mediated ectodomain shedding of EGF-like factors

Epidermal growth factor (EGF)-like proteins comprise a group of structurally similar growth factors, which contain a conserved six-cysteine residue motif called the EGF-domain. EGF-like factors are synthesized as transmembrane precursors, which can undergo proteolytic cleavage at the cell surface to release a mature soluble ectodomain; a process often referred to as “ectodomain shedding”. Ectodomain shedding of EGF-like factors has been linked to multiple zinc-binding metalloproteases of the matrix metalloprotease (MMP) and a disintegrin and metalloprotease (ADAM) families. Shedding can be activated by a variety of pharmacological and physiological stimuli and these activation events have been linked to the enhancement of metalloprotease activity, possibly via the action of intracellular signaling modules. Once shed from the cell surface, EGF-like factors bind to a family of four cell surface receptors named ErbB-1, -2, -3 and -4. Heterodimerization or homodimerization of these receptors following ligand binding drives intracellular signal transduction cascades, which eventuate in diverse cell fates including proliferation, differentiation, migration and inhibition of apoptosis. In addition to its role in driving normal developmental processes, a wealth of evidence now exists showing that de-regulated ErbB signaling is associated with the formation of tumors in a variety of tissues and that ectodomain shedding of EGF-like factors plays a critical event in this process. Thus, knowledge of the molecular mechanisms by which EGF-like factors are shed from the cell surface and the nature of the proteases and cellular signals that govern this process is crucial to understanding ErbB receptor signaling and potentially also in the development of novel cancer therapeutics targeting the ErbB pathway. This review focuses on the structure and function of EGF-like factors, and the mechanisms that govern the shedding of these transmembrane molecules from the cell surface.

Structure-function and biological role of betacellulin

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.

Ammonium phosphate in sori of Dictyostelium discoideum promotes spore dormancy through stimulation of the osmosensor ACG

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.

Cloning of rat betacellulin and characterization of its expression in the gastrointestinal tract.

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.

Evidence for a developmentally regulated prespore-specific glutamine synthetase in the cellular slime mould Dictyostelium discoideum.

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.

Betacellulin promotes growth of the gastrointestinal organs and effects a diuresis in normal rats.

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.

Identification of an Alternatively Spliced mRNA Transcript of Human Betacellulin Lacking the C-Loop of the EGF Motif and the Transmembrane Domain

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.

Identification of Betacellulin as a Major Growth Factor in Foetal Bovine Serum and Development of a Recombinant Betacellulin Analogue for Use in Serum-Free Cell Culture

Continued concerns about animal-derived raw materials have increased the requirement for alternative components that can be used in cell culture. Foetal bovine serum (FBS) contains many growth factors and bioactive proteins, which together contribute to its potency in cell culture. Identification and subsequent production of such factors by recombinant DNA methods followed by incorporation into serum-free media would be of benefit to companies and regulatory authorities. Recently, we isolated and sequenced a bovine homologue of betacellulin (bBTC), a member of the epidermal growth factor family, from bovine milk (Dunbar et al., 1999). This paper describes the production of recombinant bovine BTC, a polyclonal antiserum against bBTC and its use to demonstrate BTC as a major growth factor in FBS. We also describe the production of a BTC analogue (LongBTC) designed specifically for use in serum-free media, and its biological potency in fibroblasts.