Control of Schwann cell survival and proliferation: autocrine factors and neuregulins.

Postnatal rat Schwann cells secrete factors that prevent the programmed cell death (PCD) of low-density Schwann cells in serum-free culture. These autocrine survival signal(s) do not promote Schwann cell proliferation. Moreover, while NRG and bFGF, which promote proliferation, both rescue a subpopulation of neonatal Schwann cells from PCD, they do not rescue freshly isolated Schwann cells from older animals; other known protein factors tested also do not mimic the autocrine signal. These results suggest that Schwann cells switch their survival dependency around the time of birth from axonal signals such as NRG to autocrine signals. Such an arrangement would be advantageous for the regeneration of peripheral axons following injury. We also compared NRG-induced Schwann cell proliferation using autocrine signals or serum to promote survival. The autocrine signals increase the rate of NRG-stimulated proliferation of low-density Schwann cells in serum-free medium, whereas serum inhibits proliferation by inhibiting both the production of survival signals and the expression of erbB2 and erbB3 receptors; these inhibitions are all reversed by forskolin. In contrast, forskolin has no effect on proliferation when the cells are exposed to high levels of autocrine factors.

p120, a p120-related protein (p100), and the cadherin/catenin complex.

Cadherins and catenins play an important role in cell-cell adhesion. Two of the catenins, beta and gamma, are members of a group of proteins that contains a repeating amino acid motif originally described for the Drosophila segment polarity gene armadillo. Another member of this group is a 120-kD protein termed p120, originally identified as a substrate of the tyrosine kinase pp60src. In this paper, we show that endothelial and epithelial cells express p120 and p100, a 100-kD, p120-related protein. Peptide sequencing of p100 establishes it as highly related to p120. p120 and p100 both appear associated with the cadherin/catenin complex, but independent p120/catenin and p100/catenin complexes can be isolated. This association is shown by coimmunoprecipitation of cadherins and catenins with an anti-p120/p100 antibody, and of p120/p100 with cadherin or catenin antibodies. Immunocytochemical analysis with a p120-specific antibody reveals junctional colocalization of p120 and beta-catenin in epithelial cells. Catenins and p120/p100 also colocalize in endothelial and epithelial cells in culture and in tissue sections. The cellular content of p120/p100 and beta-catenin is similar in MDCK cells, but only approximately 20% of the p120/p100 pool associates with the cadherin/catenin complex. Our data provide further evidence for interactions among the different arm proteins and suggest that p120/p100 may participate in regulating the function of cadherins and, thereby, other processes influenced by cell-cell adhesion.

Evidence for excitoprotective and intraneuronal calcium-regulating roles for secreted forms of the beta-amyloid precursor protein.

The beta-amyloid precursor protein (beta APP) is a membrane-spanning glycoprotein that is the source of the beta-amyloid peptide (beta AP) which accumulates as senile plaques in the brains of patients with Alzheimer's disease. beta APP is normally processed such that a cleavage occurs within the beta AP, liberating secreted forms of beta APP (APPss) from the cell. The neuronal functions of these forms are unknown. We now report that APPss have a potent neuroprotective action in cultured rat hippocampal and septal neurons and in human cortical neurons. APPs695 and APPs751 protected neurons against hypoglycemic damage, and the neuroprotection was abolished by antibodies to a specific region common to both APPs695 and APPs751. APPss caused a rapid and prolonged reduction in [Ca2+]i and prevented the rise in [Ca2+]i that normally mediated hypoglycemic damage. APPss also protected neurons against glutamate neurotoxicity, effectively raising the excitotoxic threshold. APPss may normally play excitoprotective and neuromodulatory roles. Alternative processing of APPss in Alzheimer's disease may contribute to neuronal degeneration by compromising the normal function of APPss and by promoting the deposition of beta AP.

Axonal sprouting induced in the sciatic nerve by the amyloid precursor protein (APP) and other antiproteases.

Protease inhibition is the mechanism by which some trophic factors promote the extension of neurites. In the rat sciatic nerve, we assessed the ability to induce sprouts of the APP isoform that embodies the Kunitz antiprotease domain and other antiproteases. With the electron microscope, axonal sprouts were found when antiproteases were supplied but not after administration of inactive substances. We conclude that axons have a drive to sprout which can be released by the unbalance of an extracellular protease-antiprotease system. We propose that this system is involved in the pathogenesis of Alzheimer's disease.

Exact cleavage site of Alzheimer amyloid precursor in neuronal PC-12 cells.

We have identified the secretory cleavage site in the Alzheimer amyloid precursor (APP) in a non-transfected neuronal cell line, using cyanogen bromide digests of APP purified from medium conditioned by PC-12 cells which were differentiated to a neuronal phenotype. The results obtained are most consistent with proteolysis of the Lys16-Leu17 bond in the beta amyloid peptide, followed by partial removal of Lys16 by a basic carboxypeptidase.

Isolation and characterization of opioid peptides from rabbit cerebellum.

The rabbit cerebellum has been shown to contain significant quantities of opioid receptors consisting of both mu- and kappa-subtypes. To determine the nature of the endogenous opioid ligands in this tissue, extracts from rabbit cerebellum were separated by various chromatography techniques and fractions were assayed initially for opioid peptides with a radioimmunoassay capable of detecting all peptides with an amino-terminal Tyr-Gly-Gly-Phe sequence. This sequence is common to all mammalian opioid peptides and is critical for recognition by all known opioid receptors. Each of the three immunoreactive opioid peptide peaks detected was purified to homogeneity and subjected to amino acid composition and sequence analysis. One peak was analyzed further by mass spectrometry. This identified the major opioid peptides in the cerebellum as [Met5]enkephalin, [Leu5]enkephalin, and heptapeptide [Met5]enkephalyl-Arg6-Phe7. The comprehensiveness of this initial detection scheme in identifying biologically active opioid peptides was substantiated through subsequent analysis. Using specific radioimmunoassays for representative opioid peptides of the three opioid systems currently known, no other peptides of either the proenkephalin, proopiomelanocortin, or prodynorphin series were detected in any appreciable amounts. Collectively, these results are consistent with the position that rabbit cerebellar opioids are derived from proenkephalin. However, given that no appreciable quantities of either [Met5]enkephalyl-Arg6-Arg7-Val8-NH2 (metorphamide) or [Met5]enkephalyl-Arg6-Gly7-Leu8 were detected suggests that rabbit proenkephalin may have a slightly altered sequence and/or is differentially processed relative to other mammalian species studied.

Cleavage of amyloid beta peptide during constitutive processing of its precursor.

The amyloid beta peptide (A beta P) is a small fragment of the much larger, broadly distributed amyloid precursor protein (APP). Abundant A beta P deposition in the brains of patients with Alzheimer's disease suggests that altered APP processing may represent a key pathogenic event. Direct protein structural analyses showed that constitutive processing in human embryonic kidney 293 cells cleaves APP in the interior of the A beta P, thus preventing A beta P deposition. A deficiency of this processing event may ultimately prove to be the etiological event in Alzheimer's disease that gives rise to senile plaque formation.

cDNA cloning and sequencing of the protein-tyrosine kinase substrate, ezrin, reveals homology to band 4.1.

Ezrin is a component of the microvilli of intestinal epithelial cells and serves as a major cytoplasmic substrate for certain protein-tyrosine kinases. We have cloned and sequenced a human ezrin cDNA and report here the entire protein sequence derived from the nucleotide sequence of the cDNA as well as from partial direct protein sequencing. The deduced protein sequence indicates that ezrin is a highly charged protein with an overall pI of 6.1 and a calculated molecular mass of 69,000. The cDNA clone was used to survey the distribution of the ezrin transcript, and the 3.2 kb ezrin mRNA was found to be expressed in the same tissues that are known to express the protein and at the same relative levels. Highest expression was found in intestine, kidney and lung. The cDNA clone hybridized to DNAs from widely divergent organisms indicating that its sequence is highly conserved throughout evolution. The amino acid sequence of ezrin revealed a high degree of similarity within its N-terminal domain to the erythrocyte cytoskeletal protein, band 4.1 and secondary structure predictions indicate that a second region of ezrin contains a long alpha-helix, a feature also common to band 4.1. The structural similarity of ezrin to band 4.1 suggests a mechanism for the observed localization to the membrane, and a role for ezrin in modulating the association of the cortical cytoskeleton with the plasma membrane.

Structural characterization of follistatin: a novel follicle-stimulating hormone release-inhibiting polypeptide from the gonad.

Follistatin, a novel, single chain, glycosylated polypeptide bearing no homology with previously characterized inhibins but exhibiting potent and specific pituitary FSH-release inhibition has been structurally characterized by protein microsequencing, cDNA cloning, and DNA sequencing. Two populations of clones differing in their 3'-untranslated sequences were found to encode a 344 amino acid precursor protein and an identical but carboxyl terminal truncated 317 amino acid precursor, respectively. Additionally, one clone, FS18, contained two introns and probably resulted from reverse transcription of heterogeneous nuclear RNA during cDNA library construction. Follistatin is unusually cysteine-rich, containing 36 cysteines in the mature coding sequence of 315 amino acids and an extremely acidic carboxyl terminal region, FS(292-304), comprised of Glu-Asp-Thr-Glu-Glu-Glu-Glu-Glu-Asp-Glu-Asp-Gln-Asp which probably resides outside a tightly cross-linked protein sphere. The heparin-binding ability of follistatin can probably be ascribed to the basic region specified by FS(75-86), Lys-Lys-Cys-Arg-Met-Asn-Lys-Lys-Asn-Lys. Overall, follistatin is organized into three homologous domains, FS(66-135), FS(139-210), and FS(216-287) containing 70, 72, and 72 amino acids, respectively, which show a 52% homology among themselves and a 57% homology with the 56 amino acid human pancreatic secretory trypsin inhibitor protein when aligned for maximum homology.

The Mr 24,000 phosphoprotein from developing bone is the NH2-terminal propeptide of the alpha 1 chain of type I collagen.

Using nondegradative isolation procedures, we have purified and characterized the Mr 24,000 phosphoprotein from developing bovine and human bone where it constitutes 5% of the noncollagenous protein in the mineral compartment. This hydroxyproline-containing protein could not be cleaved by cyanogen bromide. The purified, intact product spontaneously formed a complex consistent with a collagen-like trimer that remained a trimer even in sodium dodecyl sulfate-polyacrylamide gels. The ability to form the complex was lost upon treatment with bacterial collagenase, a treatment that resulted in an NH2-terminally blocked fragment of Mr 17,000. After deblocking, the NH2-terminus of the intact, Mr 24,000 bovine product was shown to have virtually the same amino acid sequence (residues 1-24 with asparagine rather than aspartic acid at position 20 as reported earlier by Horlein et al. (Horlein, D., Fietzek, P. P., Wachter, E., Lapiere, C. M., and Kuhn, K. (1979) Eur. J. Biochem. 90, 31-38) as the amino-terminal segment of dermatosparatic calf skin alpha 1 type I procollagen. Furthermore, pulse-chase studies showed a precursor-product relationship between procollagen and the Mr 24,000 protein. Anti-serum made against the bovine bone protein bound to bands on electrotransfers that were consistent with the positions of both alpha 1(I) procollagen and the procollagen chain missing its COOH-terminal extension peptide (pN-alpha 1(I), as well as the original Mr 24,000 product in extracts of bone, skin, tendon, cornea, and other type I collagen-containing tissues. Fetal calf serum contained an average of 106 micrograms/ml of the Mr 24,000 protein as determined by quantitative enzyme-linked immunosorbent assay. The only serine residue in the bovine bone protein was phosphorylated. It is unknown whether the corresponding collagen NH2-terminal pro-peptides in other tissues and serum are similarly phosphorylated.

Complementary deoxyribonucleic acid (cDNA) cloning and DNA sequence analysis of rat ovarian inhibins.

Two forms of inhibin (A and B), gonadal polypeptide hormones that selectively suppress the secretion of FSH from the anterior pituitary, have been characterized from the porcine and human species, each being composed of a common alpha-chain and one of two distinct, but homologous beta-chains, i.e. alpha beta A and alpha beta B. Using cDNAs encoding the porcine inhibin subunits we have cloned and sequenced the cDNAs encoding the alpha, beta A, and beta B chains of rat ovarian inhibin. Northern analyses of rat testicular RNA with rat ovarian cDNA probes show the presence of mRNAs encoding alpha and beta B chains, but no detectable mRNA encoding the beta A chain under our experimental conditions. This suggests that there may be specific and distinct physiological roles for inhibins A and B. In addition, if there is no extratesticular source of beta A mRNA, then the male rat may be devoid of the stimulators of the secretion of FSH, i.e. activin (beta A beta B) and homoactivin A (beta A beta A), which are derived from the beta subunits of the two inhibins.

Identification of the gene encoding an RNA polymerase-binding protein of bacteriophage T4.

One of five bacteriophage T4-specified proteins that bind to host RNA polymerase core has been purified and partially sequenced. A mixed oligonucleotide, based on the amino acid sequence, was used to probe genomic restriction fragments. The gene for this protein, previously designated the 15K protein, has been located between T4 genes 45 and 46 and designated rpbA.

Cloning and sequence analysis of cDNA for bovine carboxypeptidase E.

Carboxypeptidase E (enkephalin convertase) was first identified as the carboxypeptidase B-like enzyme involved in the biosynthesis of enkephalin in bovine adrenal chromaffin granules. A similar enzyme is present in many brain regions and in purified secretory granules from rat pituitary and rat insulinoma. Within the secretory granules, carboxypeptidase E (CPE) activity is found in both a soluble and a membrane-bound form, which differ slightly in relative molecular mass (Mr). Here, to investigate whether the CPE activities in the various tissues are produced from a single gene, purified CPE was partially sequenced and oligonucleotide probes were used to isolate a clone encoding CPE from a bovine pituitary complementary DNA library. This cDNA hybridizes to bovine pituitary poly(A)+ RNAs of approximately 3.3, 2.6 and 2.1 kilobases (kb), with the 3.3-kb messenger RNA the predominant species. The predicted amino-acid sequence of the cDNA clone contains the partially determined sequences of CPE, several pairs of basic amino acids and displays some homology with both carboxypeptidases A and B. Restriction analysis of bovine genomic DNA suggests only one gene for CPE. This is consistent with a broad role for CPE in the biosynthesis of many neuropeptides.

Cloning and DNA sequence analysis of the cDNA for the precursor of the beta chain of bovine follicle stimulating hormone.

Follicle stimulating hormone (FSH) plays essential roles in the maintenance and development of oocytes and spermatozoa in normal reproductive physiology. FSH possesses two subunits, alpha and beta, the latter being responsible for FSH biological specificity. We have cloned and sequenced the cDNA encoding the FSH beta chain from a bovine anterior pituitary cDNA library. The mature molecule is 109 amino acids long and is preceded by a 20-amino acid putative signal peptide. RNA gel blot analysis of bovine pituitary RNA shows that the mRNA encoding beta chain of FSH is approximately 1.7 kilobases in length.

Isolation and characterization of an endogenous C-terminal fragment of the alpha-neo-endorphin/dynorphin precursor from bovine caudate nucleus.

Antibodies have been raised to a synthetic peptide corresponding to the C-terminal 15-amino acid residues of prodynorphin, the common precursor to the neo-endorphins and dynorphins. The amino acid sequence of the antigen was based on the sequence deduced from mRNA isolated and cloned from porcine hypothalamus (Kakidani, H., Y. Furutani, H. Takahashi, M. Noda, Y. Morimoto, T. Hirose, M. Asai, S. Inayama, S. Nakanishi, and S. Numa (1982) Nature 298: 245-248). Using a radioimmunoassay developed from these antibodies we have isolated an endogenous prodynorphin C-fragment from bovine caudate nucleus. The isolated peptide displayed characteristics on gel filtration similar to those of synthetic prodynorphin C-fragment predicted from the porcine mRNA sequence but had low cross-reactivity in the radioimmunoassay. Sequencing and amino acid analysis showed a substitution of serine for asparagine at position 6 in the porcine sequence. Dynorphin B (rimorphin), which is adjacent to prodynorphin C-fragment in the precursor, was isolated from the same extract. Amino acid analysis and elution position on a gel filtration column confirmed its structure as that previously characterized from bovine pituitary extracts. The release of prodynorphin C-fragment and the C-terminus of dynorphin B from the porcine precursor would require cleavage at a single arginine residue. However, a terminal arginine was not present on either of these prodynorphin peptides isolated from bovine caudate. The data would suggest that processing at a single arginine residue results in elimination of the arginine, a feature in common with processing at paired basic residues.

Isolation and characterization of epidermal growth factor from human milk.

Epidermal growth factor (EGF) has been purified from human milk. The purification was monitored with a human placental membrane radioreceptor assay using murine salivary epidermal growth factor I (mEGF I) as a competitive ligand and was achieved exclusively by the use of reverse-phase liquid chromatography (RPLC). The sequential use of preparative, semipreparative and analytical RPLC on an octylsilica support with solvent systems of different solute selectivity such as pyridine formate, triethylammonnium phosphate or perfluorocarbonic acids in the presence of n-propanol or acetonitrile allowed purification to homogeneity with 5 consecutive runs. The molecular mass, amino acid composition and NH2-terminal sequence of human EGF were determined. Gas-phase microsequencing of residues 1-17 revealed the following sequence: Asn-Ser-Asp-Ser-Glu-X-Pro-Leu-Ser-His-Asp-Gly-Tyr-X-Leu-X-Asp which is identical with the NH2-terminof urogastrone from human urine. The purified polypeptide competes with mEGF for the placental membrane receptor with a ki of 1 ng. Furthermore, it stimulates the anchorage-dependent as well as -independent proliferation of human and rat indicator cells with half-maximal stimulation at 1 and 2.5 ng/ml, respectively. Although human epidermal growth factor has been unequivocally identified in human milk and -for the first time-shown to be identical with urogastrone from human urine, the high-resolution techniques employed have also revealed the presence of EGF-related molecules which await further characterization. It is possible that EGF and the EGF-related growth factors possess important regulatory functions in normal growth of the human breast during pregnancy and lactation as well as in abnormal growth during mammary tumor formation and progression.

Characterization of a Saponaria officinalis seed ribosome-inactivating protein: immunoreactivity and sequence homologies.

A ribosome inactivating protein from Saponaria officinalis, SO-6, was purified and the N-terminus sequenced. The sequence shows extensive homology with Pokeweed antiviral protein, Pokeweed antiviral protein II, Pokeweed antiviral seed protein and dodecandrin. SDS gel electrophoresis in the Laemmli system revealed two bands of similar intensities with a smear between them, probably an artifact due to the high pI of the protein. Use of a harsher denaturing gel system resulted in one band in electrophoresis. Immune antisera was raised in rabbits against this protein and it cross reacted with other proteins (SO-5, SO-8 and SO-9) from seeds of Saponaria officinalis, but not with gelonin, Momordica charantia inhibitor and dianthin 32.

Isolation and structure of a novel C-terminally amidated opioid peptide, amidorphin, from bovine adrenal medulla.

Biologically active peptide hormones and neurotransmitters have been shown to be enzymatically liberated from larger, inactive precursor molecules by tissue-specific post-translational processing, particularly at the typical cleavage signals of paired basic residues. Subsequent N-terminal or C-terminal modifications may be of importance in regulating the biological activities of these peptides. C-terminal alpha-amidation is considered to be essential for the biological function of several non-opioid peptides. Here we present the isolation and structure of a novel C-terminally amidated opioid peptide, amidorphin, from bovine adrenal medulla. Amidorphin and the recently isolated octapeptide metorphamide (adrenorphin) are the only endogenous opioid peptides in mammals known to possess a C-terminal amide group. The amino acid sequence of amidorphin corresponds to the sequence 104-129 of bovine proenkephalin A. Very high concentrations of amidorphin were detected in bovine adrenal medulla and in a further endocrinological system, the hypothalamic-neurohypophyseal axis. Amidorphin may therefore be considered to be a major gene product of the opioid peptide precursor proenkephalin A in these endocrine tissues.

Phosphorylation sites in enolase and lactate dehydrogenase utilized by tyrosine protein kinases in vivo and in vitro.

Enolase, lactate dehydrogenase, and phosphoglycerate mutase have previously been found to contain phosphotyrosine in fibroblasts transformed by Rous sarcoma virus, which encodes a tyrosine-specific protein kinase. However, these phosphorylations are not stoichiometric, and their significance for any aspect of the transformed phenotype is unknown. We show here that enolase and lactate dehydrogenase are each phosphorylated chiefly at a single tyrosine in Rous sarcoma virus-transformed cells. The purified enzymes can also be phosphorylated at the same tyrosine in vitro when incubated with an immunoprecipitated retroviral transforming protein having associated tyrosine protein kinase activity. The phosphorylated tyrosine in lactate dehydrogenase is amino acid 238. The phosphorylated tyrosine in enolase lies in a sequence homologous to that surrounding histidine 43 in yeast enolase. Although the phosphorylated sequence in lactate dehydrogenase shows some homology to those sequences surrounding phosphotyrosines found in retroviral transforming proteins, the phosphorylated sequence in enolase is quite different.

Growth hormone-releasing factor regulates growth hormone mRNA in primary cultures of rat pituitary cells.

A peptide with high intrinsic activity for specifically stimulating the secretion of immunoreactive growth hormone (GH; somatotropin) has been characterized and reproduced by total synthesis. This peptide, human pancreatic growth hormone-releasing factor, 44-amino-acid form (hpGRF1-44-NH2), was isolated from a tumor localized in the pancreas of a patient with acromegaly. We report here the effect of this growth hormone-releasing factor (GRF) on GH release and the GH mRNA levels in monolayer cultures of rat pituitary. The cytoplasmic dot hybridization technique was used to examine the effect of GRF on GH mRNA levels. Incubation of rat pituitary cultures with GRF for 72 hr resulted in a 2- to 2.5-fold increase in GH mRNA levels, and the maximal levels of stimulation were achieved at GRF concentrations as low as 1 fM. GRF did not stimulate prolactin release, nor did it affect specific prolactin mRNA levels in the pituitary cultures. We conclude that GRF is a potent and specific GH secretagogue that also affects specifically GH mRNA levels in normal pituitary cells.