Three neurotoxins from the venom of a sea snake Astrotia stokesii, including two long-chain neurotoxic proteins with amidated C-termini.

From the venom of a sea snake Astrotia stokesii three neurotoxic components, toxins Astrotia stokesii a, b and c were isolated in 40, 15 and 5% yield by weight respectively of the whole venom. Their LD50 values for 20g mice were 0.13, 0.096 and 0.098 microgram/g body wt. respectively and accounted for almost all the lethal activity of the venom. Their amino acid sequences were determined. Astrotia stokesii a was composed of 60 amino acid residues with nine half-cystine residues and was quite homologous to other sea-snake short-chain neurotoxins in its amino acid sequence. Toxins Astrotia stokesii b and c were composed of 70 and 72 amino acid residues respectively with 10 half-cystine residues. They are the first long-chain neurotoxins with high activity isolated from sea-snake venoms. The C-terminal carboxy groups of toxins b and c were found to be amidated; the amidation is known for some polypeptides, but is novel for a protein. The amide group may make a hydrogen-bond with glutamic acid-39, which replaces a lysine that has so far been found invariably in long-chain neutrotoxins. Astrotia stokesii b and c are also novel in having phenylalanine-25 and isoleucine- or valine-42. The ordinary Tyr-Glu pair, which is observed in X-ray structure [Low, Preston, Sato, Rosen, Searl, Rudko & Richardson (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2991-2994] and n.m.r.study [Inagaki, Tatsumi, Miyazawa, Hori & Tamiya (1977) Abstr. Int. Congr. Pure Appl. Chem. 26th, p. 336] on erabutoxins may be replaced by a hydrophobic pair. Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication SUP 5009o (30 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7B1, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.     

Electrogenic Na/HCO3 cotransporter (NBCe1) variants expressed in Xenopus oocytes: functional comparison and roles of the amino and carboxy termini

Using pH- and voltage-sensitive microelectrodes, as well as the two-electrode voltage-clamp and macropatch techniques, we compared the functional properties of the three NBCe1 variants (NBCe1-A, -B, and -C) with different amino and/or carboxy termini expressed in Xenopus laevis oocytes. Oocytes expressing rat brain NBCe1-B and exposed to a CO(2)/HCO(3)(-) solution displayed all the hallmarks of an electrogenic Na(+)/HCO(3)(-) cotransporter: (a) a DIDS-sensitive pH(i) recovery following the initial CO(2)-induced acidification, (b) an instantaneous hyperpolarization, and (c) an instantaneous Na(+)-dependent outward current under voltage-clamp conditions (-60 mV). All three variants had similar external HCO(3)(-) dependencies (apparent K(M) of 4-6 mM) and external Na(+) dependencies (apparent K(M) of 21-36 mM), as well as similar voltage dependencies. However, voltage-clamped oocytes (-60 mV) expressing NBCe1-A exhibited peak HCO(3)(-)-stimulated NBC currents that were 4.3-fold larger than the currents seen in oocytes expressing the most dissimilar C variant. Larger NBCe1-A currents were also observed in current-voltage relationships. Plasma membrane expression levels as assessed by single oocyte chemiluminescence with hemagglutinin-tagged NBCs were similar for the three variants. In whole-cell experiments (V(m) = -60 mV), removing the unique amino terminus of NBCe1-A reduced the mean HCO(3)(-)-induced NBC current 55%, whereas removing the different amino terminus of NBCe1-C increased the mean NBC current 2.7-fold. A similar pattern was observed in macropatch experiments. Thus, the unique amino terminus of NBCe1-A stimulates transporter activity, whereas the different amino terminus of the B and C variants inhibits activity. One or more cytosolic factors may also contribute to NBCe1 activity based on discrepancies between macropatch and whole-cell currents. While the amino termini influence transporter function, the carboxy termini influence plasma membrane expression. Removing the entire cytosolic carboxy terminus of NBCe1-C, or the different carboxy terminus of the A/B variants, causes a loss of NBC activity due to low expression at the plasma membrane.     

A Targeted Mutation within the Feline Leukemia Virus (FeLV) Envelope Protein Immunosuppressive Domain To Improve a Canarypox Virus-Vectored FeLV Vaccine

We previously delineated a highly conserved immunosuppressive (IS) domain within murine and primate retroviral envelope proteins that is critical for virus propagation in vivo. The envelope-mediated immunosuppression was assessed by the ability of the proteins, when expressed by allogeneic tumor cells normally rejected by engrafted mice, to allow these cells to escape, at least transiently, immune rejection. Using this approach, we identified key residues whose mutation (i) specifically abolishes immunosuppressive activity without affecting the “mechanical” function of the envelope protein and (ii) significantly enhances humoral and cellular immune responses elicited against the virus. The objective of this work was to study the immunosuppressive activity of the envelope protein (p15E) of feline leukemia virus (FeLV) and evaluate the effect of its abolition on the efficacy of a vaccine against FeLV. Here we demonstrate that the FeLV envelope protein is immunosuppressive in vivo and that this immunosuppressive activity can be “switched off” by targeted mutation of a specific amino acid. As a result of the introduction of the mutated envelope sequence into a previously well characterized canarypox virus-vectored vaccine (ALVAC-FeLV), the frequency of vaccine-induced FeLV-specific gamma interferon (IFN-γ)-producing cells was increased, whereas conversely, the frequency of vaccine-induced FeLV-specific interleukin-10 (IL-10)-producing cells was reduced. This shift in the IFN-γ/IL-10 response was associated with a higher efficacy of ALVAC-FeLV against FeLV infection. This study demonstrates that FeLV p15E is immunosuppressive in vivo, that the immunosuppressive domain of p15E can modulate the FeLV-specific immune response, and that the efficacy of FeLV vaccines can be enhanced by inhibiting the immunosuppressive activity of the IS domain through an appropriate mutation.     

Epidermal growth factor (EGF) increases the renal amino acid transport capacity in amino acid loaded rats

Summary In anaesthetized adult female rats, the influence of epidermal growth factor (EGF) on renal amino acid handling was investigated in glutamine, arginine (both 50 mg/100 g b. wt. per hour), or alanine (90 mg/ 100 g b. wt. per hour) loaded animals. Continuous infusions of the three amino acids were followed by an increase in the fractional excretion (FE) of the administered amino acids as well as of the other endogenous amino acids. Under load conditions (alanine, arginine or glutamine), EGF pretreatment (8g/100g b. wt. subcutaneously for 8 days, twice daily 8 a.m. and 4 p.m.) was followed by a stimulation of renal amino acid reabsorption. The increase in the fractional excretion of the administered amino acids was significantly lower than in non-EGF-treated rats. These changes in amino acid transport were connected with a significant reduction of GFR after EGF pretreatment (0.96 ± 0.10 vs. 0.62 ± 0.07 ml/min X 100 g b. wt.) and a distinct increase in sodium excretion (2.98 ± 0.55 vs. 4.97 ± 0.71val/100 g b. wt. X 20 min). After loading with p-aminohippurate (PAH; 200mg/100g b. wt.), PAH excretion in EGF rats was increased by about 20%, whereas urinary protein excretion was lower in EGF pretreated rats (control: 0.45 ± 0.04 vs. EGF: 0.18 ± 0.03 mg/ 100 g b. wt. X 20 min). The PAH load reduced amino acid reabsorption as a sign of overloading of renal tubular transport capacity, but in EGF pretreated animals the amino acid excretion was only slightly increased under these conditions. Furthermore, EGF pretreatment depressed normal kidney weight gain significantly (874 ± 18 vs. 775 ± 32mg/100g b. wt.). EGF can improve the renal tubular transport capacity, but, compared to well-known stimulators of renal transport like dexamethasone or tri-iodothyronine, its effect is only of a moderate degree.     

Carboxypeptidase activity in synaptic vesicles isolated from striatum and cortex of calf brain

A carboxypeptidase activity has been found in synaptic vesicles (secretory granules) isolated from the cortex and striatum of calf brain which removes amino acids from the carboxy terminus of enkephalin-containing (EC) peptides. The formed enkephalin molecules are not further degraded by this enzyme activity. The preparations were found to be free of cytoplasmic and lysosomal constituents as determined by marker enzyme activities. The vesicle preparations of both cortex and striatum showed differences in the degradation velocities of the various EC peptides depending on size and charge of the amino acid present at the carboxy terminus. The pH optimum of the release of Met-enkephalin from Met-enkephalin-Arg6 has been shown to be between pH 5 and 6. The enzyme activity is inhibited by thiol-blocking agents such as p-hydroxymercuribenzoate and copper ions, but only slightly by metal-chelating agents.     

Inhibition of EGF processing in responsive and nonresponsive human fibroblasts

We have examined the proteolytic processing of radiolabeled epidermal growth factor (EGF) in EGF growth-responsive human foreskin fibroblasts (HFF) versus EGF nonresponsive human fetal lung fibroblasts (HFL). Previous studies (Schaudies et al., 1985) have shown that both cell lines demonstrate similar binding affinities and numbers of binding sites, as well as similar rates of internalization and degradation of the bound, radiolabeled hormone. We have used nondenaturing electrophoresis to compare how these two cell lines process EGF at its carboxy terminus. EGF lacking either one [des-(53)-EGF] or six [des (48-53)-EGF] carboxy terminal amino acids could be distinguished by this method. Chloroquine or leupeptin were added to the incubation system in an attempt to accentuate potential differences in hormonal processing between the responsive and nonresponsive cell lines. In the absence of inhibitors, the responsive and nonresponsive cells generated similar distributions of processed forms of EGF after 30-minutes incubation. However, after 4-hours incubation in the constant presence of 125I-EGF, the electrophoretic profiles of extracted hormone were substantially different. The radiolabel within the responsive cells, as well as that released from them, migrated predominantly at the dye front, indicating complete degradation of EGF. In contrast, the majority of the radiolabel within the nonresponsive cells migrated as partially processed forms of hormone, while the released radiolabel migrated at the dye front. Addition of chloroquine to either cell line inhibited processing of EGF beyond removal of the carboxyl terminal arginine residue. Both intact 125I-EGF, and 125I-EGF lacking the carboxyl terminal arginine were released from chloroquine-treated cells in a ratio equal to that present in the intact cells. Incubations in leupeptin, proteolysis of EGF beyond the des-(48-53)-EGF was blocked; however, no large-molecular-weight species were released from the cells under these conditions.     

Immune response to ferredoxin; nonresponder status is not due to suppression

Ferredoxin (Fd), a small protein from Clostridium pasteurianum, has been selected for immunologic studies because of its limited number (two) of antigenic determinants. Functionally (as determined by antibody binding), monodeterminant fragments of Fd can be generated enzymatically, leaving molecules only a few amino acids smaller than the native protein, with unaltered solid phase binding properties. These fragments were used to assess the immune response to each of the two determinants. Clear differences in immunologic properties can be assigned to sequences within Fd: the amino terminal tripeptide is responsible for inducing a proliferative response and limited antibody production, whereas the carboxy terminal dipeptide accounts for most of the antibody activity, yet little, if any, T-proliferative activity. Studies with the enzyme-generated fragments of Fd have unmasked a sequence proximal to the amino terminal that represents a second determinant for T cell proliferation but does not have any demonstrable antibody-inducing activity. This third determinant is shown to induce responsiveness to Fd in nonresponder animals after the removal of the amino terminal tripeptide. The results indicate that nonresponsiveness to this molecule in H-2d mice is not a direct effect of suppression.     

Structural similarities among enzyme pterin binding sites as demonstrated by a monoclonal anti-idiotypic antibody

BALB/c mice were immunized with a synthetic co-factor of the aromatic amino acid hydroxylases, 6,7-dimethyl-5,6,7,8-tetrahydropterin, conjugated to albumin. Hybridoma cell lines isolated from the immunized mice secreted monoclonal antibodies reacting specifically with the pterin molecule and monoclonal antibodies which were found to bind phenylalanine hydroxylase. Several lines of evidence were consistent with the anti-phenylalanine hydroxylase antibodies being anti-idiotype antibodies mimicking the pterin molecule and binding to the pterin binding site of phenylalanine hydroxylase. (a) An anti-idiotype monoclonal antibody, NS7, when reimmunized into mice produced anti-pterin antibodies consistent with NS7 being an internal image anti-idiotypic antibody. (b) NS7 antibody was prevented from binding to phenylalanine hydroxylase when a competitive inhibitor of phenylalanine hydroxylase enzyme activity, 6,7-dimethyl-7,8-dihydropterin, was bound to phenylalanine hydroxylase. (c) NS7 antibody was shown to bind to a wide range of pterin-requiring enzymes: phenylalanine, tyrosine and tryptophan hydroxylases, dihydropteridine reductase, dihydrofolate reductase, and sepiapterin reductase. Thus the NS7 antibody has successfully mimicked a common portion of the pterin cofactors utilized by these enzymes and demonstrated structure homology in their pterin binding sites despite their diverse function and little amino acid sequence homology except among the three aromatic amino acid hydroxylases.     

Activated Cdc42-associated kinase 1 is a component of EGF receptor signaling complex and regulates EGF receptor degradation

Cdc42-associated tyrosine kinase 1 (ACK1) is a specific down-stream effector of Cdc42, a Rho family small G-protein. Previous studies have shown that ACK1 interacts with clathrin heavy chain and is involved in clathrin-coated vesicle endocytosis. Here we report that ACK1 interacted with epidermal growth factor receptor (EGFR) upon EGF stimulation via a region at carboxy terminus that is highly homologous to Gene-33/Mig-6/RALT. The interaction of ACK1 with EGFR was dependent on the kinase activity or tyrosine phosphorylation of EGFR. Immunofluorescent staining using anti-EGFR and GFP-ACK1 indicates that ACK1 was colocalized with EGFR on EEA-1 positive vesicles upon EGF stimulation. Suppression of the expression of ACK1 by ACK-RNAi inhibited ligand-induced degradation of EGFR upon EGF stimulation, suggesting that ACK1 plays an important role in regulation of EGFR degradation in cells. Furthermore, we identified ACK1 as an ubiquitin-binding protein. Through an ubiquitin-association (Uba) domain at the carboxy terminus, ACK1 binds to both poly- and mono-ubiquitin. Overexpression of the Uba domain-deletion mutant of ACK1 blocked the ligand-dependent degradation of EGFR, suggesting that ACK1 regulates EGFR degradation via its Uba domain. Taken together, our studies suggest that ACK1 senses signal of EGF and regulates ligand-induced degradation of EGFR.     

A testis specific isoform of endophilin B1, endophilin B1t, interacts specifically with protein phosphatase-1c gamma2 in mouse testis and is abnormally expressed in PP1c gamma null mice

Male mice homozygous for a null mutation in the protein phosphatase-1c gamma (PP1c gamma) gene are infertile, displaying a severe impairment in spermatogenesis that is not compensated by the presence of PP1c alpha and PP1c beta in mutant testes. A lack of the PP1c gamma2 splice variant seems the most likely cause of the mutant phenotype, as it is the most heavily expressed PP1c gamma isoform in wild type testes. Yeast two-hybrid screening using PP1c gamma2 has identified several new binding partners, including endophilin B1t, a testis enriched isoform of endophilin B1a which differs from the somatic form by virtue of a carboxy terminal deletion spanning the last 10 amino acids. The testis isoform did not show an interaction with PP1c alpha, or with a truncated PP1c gamma2 mutant lacking the unique carboxy terminus. In contrast, somatic endophilin B1a did not interact with any of the PP1c isoforms. Sedimentation and co-immunoprecipitation experiments using native testis proteins verified binding of endophilin B1t to PP1c gamma2. Immunohistochemistry on wild type testis sections revealed a stage specific expression pattern for endophilin that appeared concentrated at discrete puncta throughout the seminiferous epithelium. Punctate endophilin expression in cells adjacent to the lumen was absent in PP1c gamma null mice. Phosphatase assays indicate that chimeric endophilin B1t is able to inhibit recombinant PP1c gamma2 activity toward phosphorylase a while having little effect on the activity of PP1c alpha. A potential role for endophilin B1t in mammalian spermatogenesis is discussed within the context of the PP1c gamma knockout testis phenotype.     

A thermoacidophilic endoglucanase (CelB) from Alicyclobacillus acidocaldarius displays high sequence similarity to arabinofuranosidases belonging to family 51 of glycoside hydrolases.

A 100-kDa protein with endoglucanase activity was purified from Triton X-100 extract of cells of the thermoacidophilic Gram-positive bacterium Alicyclobacillus acidocaldarius. The enzyme exhibited activity towards carboxy methyl cellulose and oat spelt xylan with pH and temperature optima of 4 and 80 degrees C, respectively. Cloning and nucleotide sequence analysis of the corresponding gene (celB) revealed an ORF encoding a preprotein of 959 amino acids which is consistent with an extracellular localization. Purified recombinant CelB and a variant lacking the C-terminal 203 amino acid residues (CelBtrunc) displayed similar enzymatic properties as the wild-type protein. Analysis of product formation suggested an endo mode of action. Remarkable stability was observed at pH values between 1 and 7 and 60% of activity were retained after incubation for 1 h at 80 degrees C. CelB displayed homology to members of glycoside hydrolase family 51, being only the second entry with activity typical of an endoglucanase but lacking activity on p-nitrophenyl-alpha-l-arabinofuranoside (pNPAraf). Highest sequence similarity was found towards the other endoglucanase F from Fibrobacter succinogenes (EGF), forming a distinct group in the phylogenetic tree of this family. Analysis of the amino acid composition of the catalytic domains demonstrated that CelB contains fewer charged amino acids than its neutrophilic counterparts, which is in line with adaptation to low pH. Wild-type and full-length recombinant CelB were soluble only in Triton X-100. In contrast, CelBtrunc was completely water soluble, suggesting a role of the C-terminal region in cell association. This C-terminal hydrophobic region displayed local sequence similarities to an alpha-amylase from the same organism.     

Resistance to receptor-mediated degradation of a murine epidermal growth factor analogue (EGF-Val-47) potentiates its mitogenic activity

In most cell types two classes of epidermal growth factor (EGF) receptors can be found: a major class that binds EGF with relatively low affinity and a minor class that binds with very high affinity. Structure-function studies have shown that mutations at amino acid 47 in the EGF molecule severely reduce its affinity for the EGF receptor but do not cause preferential binding to one or the other subclass of receptors. Using three EGF derivatives with a mutation at amino acid 47 (Ser-47, Leu-37-Tyr-47, and Val-47), we have investigated the relative contribution of the two receptor subclasses to the EGF-dependent mitogenic response. We show that mitogenicity correlates exclusively with occupancy of the high-affinity receptor and that full occupancy of this subclass is required for maximal stimulation. In addition we demonstrate that for the EGF-Val-47 analogue this requirement can be abrogated and half-maximal biological activity reached with a high-affinity receptor occupancy of only 8%. While the rate of internalization did not significantly differ between EGF-Val-47 and native mEGF, the analogue was much more resistant to degradation by cellular proteases and, after binding and receptor-mediated internalization, was released into the medium predominantly in an intact form. We propose that the increased mitogenicity of EGF-Val-47 is due to its prolonged half-life, resulting in continued occupancy of the high-affinity EGF receptor.     

Immunoregulatory properties of fractions from human pregnancy urine: evidence that human chorionic gonadotropin is not responsible.

The immunologically privileged position of the histoin-compatible fetus and placenta is a striking example of a physiologic immunoregulatory mechanism. This study was designed to examine the effects of human chorionic gonadotropin (HCG) on the recognitive proliferative phase and the cytotoxic effector phase of in vitro cell-mediated immune responsiveness, since HCG has previously been reported to be immunosuppressive in vitro and in vivo. Commercial preparations of HCG were found to be potent inhibitors of lymphocyte proliferative responses to nonspecific mitogens like phytohemagglutinin (PHA), specific antigens such as streptolysin-O (SLO), and allogeneic cells as measured in the one-way mixed leukocyte response. Cytotoxic effector function of lymphocytes as measured by antibody-dependent cellular cytotoxicity (ADCC) and mitogen-induced cellular cytotoxicity were also markedly inhibited by these preparations. However, the 50% inhibitory concentration varied widely from lot to lot of these commercial materials. After dialysis, a portion of the inhibitory activity was lost from some but not all HCG lots. The dialysate from those lots with diminished activity was found to be immunosuppressive in vitro but contained no HCG detectable by radioimmunoassay. Following dialysis, the immunosuppressive activity of the various HCG lots remained variable and correlated poorly with values for HCG obtained by a double antibody radioimmunoassay. HCG preparations purified to a homogeneity sufficient for amino acid sequence were found to be only minimally immunosuppressive to the in vitro PHA response and had almost no effect on proliferative responses to antigens and allogeneic cells. These data do not support the concept of a primary immunoregulatory role for HCG, but they suggest that other uncharacterized compounds partially co-purified from pregnant urine along with HCG may have such immunoregulatory activity. Further characterization and identification of this immunoregulatory material(s) is essential, since it appears to have many of the properties of an ideal immunosuppressive compound: a) nontoxicity, b) ready reversibility, c) activity at very low concentration, and d) activity on a broad range of cellular immune functions.     

In vivo immune response suppression by the supernatant from concanavalin A-activated spleen cells.

Supernatants from concanavalin A- (Con A) activated murine spleen cells have been shown to suppress the in vitro plaque-forming cell (PFC) response to sheep red blood cells (SRBC). The present study examined the effect of such Con A-activated spleen cell supernatants (herein termed CONS) on the in vivo immune response to SRBC in C57BL/6, BALB/c and CDF1 mice. CONS derived from BALB/c spleen cells suppressed direct PFC 4 and 8 days after immunization with 2 X 10(8) SRBC. CONS also suppressed indirect PFC 8 days after immunization, as well as serum hemagglutinins to SRBC. The PFC response of C57BL/6 (H-2b) mice was suppressed as much as that of BALB/c (H-2d) by CONS derived from BALB/c mice, indicating a lack of H-2 specificity of the CONS. In addition to suppression of the antibody response to SRBC, in vivo CONS administration resulted in reduction in spleen cell number. This reduction was not sufficient to explain the decreased PFC response. When the CONS was separated into less than 10,000 m.w. and greater than or equal to 10,000 m.w. fractions, the immunosuppressive activity was found in the less than 10,000 m.w. fraction. This observation suggests that intact interferon, SIRS, and MIF were not responsible for the results obtained.     

Murine epidermal growth factor: heterogeneity on high resolution ion-exchange chromatography

We have shown that epidermal growth factor (EGF) purified either by the classical method of Savage and Cohen, or solely by h.p.l.c. techniques can be resolved into two species, EGF alpha and EGF beta. However, despite the apparent purity of such materials, as determined both chromatographically and by amino acid analysis, they failed to give homogeneous products on radioiodination. Analysis by isoelectric focusing on agarose gels followed by transfer to nitrocellulose and silver staining showed that EGF alpha could be further resolved into three sub-species which focused at pH 4.6, 4.3 and 4.1. EGF beta (which also focused at pH 4.6) contained very small amounts of the species with isoelectric points of 4.1 and 4.3, probably due to slight contamination of this preparation by EGF alpha. Preparative separation of the sub-species of EGF alpha was achieved by high performance anion-exchange chromatography at pH 6.5 on a Pharmacia Mono Q column. Radioiodination of these purified sub-species did not produce significant charge heterogeneity. However, two slightly different forms of [125I]EGF alpha 1 (pH 4.6 species) were separable by anion-exchange chromatography on the Mono Q column. All of the EGF species competed for binding to EGF receptors on A431 cells and were active mitogens for BALB/c 3T3 fibroblasts.     

Murine epidermal growth factor: structure and function

Murine epidermal growth factor (EGF), a 53 amino acid protein, has been modified by enzymic digestion, site-specific chemical reactions, and recombinant DNA technology. After trypsin digestion the EGF derivatives EGF1-48 (called EGF-T) and EGF1-45 (called EGF-T2) were separated from the residual EGF and the C-terminal pentapeptide by reversed-phase high-performance liquid chromatography. EGF-T competes for binding to EGF receptors with the same efficiency as EGF. The EGF-T2 derivative had no detectable receptor binding activity even at 100 nM. The in vitro mitogenic potencies of EGF and EGF-T for Balb/c 3T3 cells were indistinguishable. Treatment of EGF-T with carboxypeptidase Y yielded two derivatives, EGF-T-(des-Arg48) and EGF-T-des(Leu47-Arg48). There was only a 3-7-fold diminution in the binding efficiency and mitogenic potency for EGF-T-(des-Arg48). However, there was more than a 100-fold decrease in the binding efficiency and mitogenic activity of EGF-T-des (Leu47-Arg48). These results indicated that Leu47 is intimately involved in the formation of the ligand-receptor complex. Studies with a number of proteases indicated that the C-terminus of EGF was susceptible to enzymic digestion; however, the N-terminus appears to be folded into a conformation which prevents access to proteolytic digestion. Consequently, the N-terminus was modified by preparing an analogue with recombinant DNA technology. Oligonucleotides corresponding to EGF(3-48). Met3 Lys21 residues were ligated in frame to a beta-galactosidase expression vector. The beta-Gal-EGF fusion protein was cleaved with cyanogen bromide and EGF(4-48).Lys21 purified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for epidermal growth factor (EGF)-induced intermolecular autophosphorylation of the EGF receptors in living cells.

In response to epidermal growth factor (EGF) stimulation, the intrinsic protein tyrosine kinase of EGF receptor is activated, leading to tyrosine phosphorylation of several cellular substrate proteins, including the EGF receptor molecule itself. To test the mechanism of EGF receptor autophosphorylation in living cells, we established transfected cell lines coexpressing a kinase-negative point mutant of EGF receptor (K721A) with an active EGF receptor mutant lacking 63 amino acids from its carboxy terminus. The addition of EGF to these cells caused tyrosine phosphorylation of the kinase-negative mutant by the active receptor molecule, demonstrating EGF receptor cross-phosphorylation in living cells. After internalization the kinase-negative mutant and CD63 have separate trafficking pathways. This limits their association and the extent of cross-phosphorylation of K721A by CD63. The coexpression of the kinase-negative mutant together with active EGF receptors in the same cells suppressed the mitogenic response toward EGF as compared with that in cells that express active receptors alone. The presence of the kinase-negative mutant functions as a negative dominant mutation suppressing the response of active EGF receptors, probably by interfering with EGF-induced signal transduction. It appears, therefore, that crucial events of signal transduction occur before K721A and active EGF receptors are separated by their different endocytic itineraries.     

Clathrin light chain: importance of the conserved carboxy terminal domain to function in living cells

Clathrin triskelions assemble into coats capable of packaging membrane and receptors for transport to intracellular destinations. A triskelion is formed from three heavy chains bound to three light chains. All clathrin light chains (clc) contain an acidic amino terminal domain, a central coiled segment, and a carboxy terminal domain conserved in amino acid sequence. To assess their functional contribution in vivo, we expressed tagged segments of the Dictyostelium clcA in clc-minus Dictyostelium (clc null) cells. We examined the ability of these clcA fragments to rescue clathrin phenotypic deficiencies, to cluster into punctae on membranes, and to bind to the heavy chain. When expressed in clc null cells, a clcA fragment containing the amino terminal domain and the central coiled domain bound heavy chain but was dispensable for clathrin function. Instead, the carboxy terminal domain of clcA was a critical determinant for association with punctae, for clathrin function and for robust binding to the heavy chain. A 70 amino acid carboxy terminal fragment was necessary and sufficient for full function, and for localization into punctae on intracellular membranes. A shorter 49 amino acid carboxy terminal fragment could distribute into punctae but failed to rescue developmental deficiencies. These results reveal the importance of the carboxy terminal domain of the light chain in vivo.