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The hallmark of the annexin super family of proteins is Ca2+-dependent binding to phospholipid bilayers, a property that resides in the conserved core domain of these proteins. Despite the structural similarity between the core domains, studies reported herein showed that annexins A1, A2, A5, and B12 could be divided into two groups with distinctively different Ca2+-dependent membrane-binding properties. The division correlates with the ability of the annexins to form Ca2+-dependent membrane-bound trimers. Site-directed spin-labeling and Forster resonance energy transfer experimental approaches confirmed the well-known ability of annexins A5 and B12 to form trimers, but neither method detected self-association of annexin A1 or A2 on bilayers. Studies of chimeras in which the N-terminal and core domains of annexins A2 and A5 were swapped showed that trimer formation was mediated by the core domain. The trimer-forming annexin A5 and B12 group had the following Ca2+-dependent membrane-binding properties: (1...

The epidermal growth factor receptor (EGFR) plays a critical role in normal growth and its overexpression is associated with several types of cancer. To learn more about regulation of the expression of this important receptor, we investigated the role of the TAFII250 subunit of transcription factor IID in the transcription of the EGFR gene. The EGFR gene has a TATA-less promoter and TAFII250 has previously been shown to have an important regulatory role in such promoters. The study was performed in the ts13 hamster cell line which has a temperature-sensitive mutation in the CCG1 gene that encodes TAFII250. At the nonpermissive temperature, the transcription of a few cell cycle-dependent genes is depressed in ts13 cells while global RNA synthesis is unaffected. Using this model system, we found that EGFR promoter-driven luciferase expression in transiently transfected ts13 cells decreased 8, 25, and 50-fold after 12, 24, and 48 hours, respectively, at the nonpermissive temperature. The decrease was partially rescued by cotransfection with the wild-type CCG1 gene. The expression of endogenous EGFR also appeared to be regulated by TAFII250—the maximum binding capacity of ts13 cells for 125I-labeled EGF decreased approximately twofold when incubated for 2 days at the nonpermissive temperature. Placing these studies in the context of the current understanding of the TFIID transcription complex, we speculate that selective stimulation of EGFR gene transcription may be mediated by TAFII250 interaction with enhancer-bound activators and the basal transcription machinery. J. Cell. Physiol. 176:642–647,1998. © 1998 Wiley-Liss, Inc.

Patients with hypohidrotic ectodermal dysplasia (HED) and Tabby (Ta) mice lack sweat glands and there is compelling evidence that these phenotypes are caused by mutations in the same highly conserved but unidentified X-linked gene. Previous studies showed that exogenous epidermal growth factor (EGF) reversed the Ta phenotype but the EGF status in HED patients has not been studied at all. Studies reported herein investigated the hypothesis that the EGF signaling pathway is involved in HED/Ta. Fibroblasts from HED patients had a two- to eightfold decrease in binding capacity for (125)I-labeled EGF, a decreased expression of the immunoreactive 170-kD EGF receptor (EGFR) protein, and a corresponding reduction in EGFR mRNA. Reduced expression of the EGFR also was observed in Ta fibroblasts and liver membranes. Other aspects of the EGF signaling pathway, including EGF concentration in urine and plasma, were normal in both HED patients and Ta mice. We propose that a decreased expression of the EGFR plays a causal role in the HED/Ta phenotype.

The annexin family of proteins is characterized by a conserved core domain that binds to phospholipids in a Ca(2+)-dependent manner. Each annexin also has a structurally distinct N-terminal domain that may impart functional specificity. To search for cellular proteins that interact with the N-terminal domain of annexin I, we constructed a fusion protein consisting of glutathione S-transferase fused to amino acids 2-47 of human annexin I (GST-AINT; AINT = annexin I N-terminal). Extracts from metabolically labeled A431 cells contained a single protein (M(r) approximately 10,000) that bound to GST-AINT in a Ca(2+)-dependent manner. A synthetic peptide corresponding to amino acids 2-18 of annexin I inhibited the binding of the 10-kDa protein to GST-AINT with half-maximal inhibition occurring at approximately 15 microM peptide. In cellular extracts, endogenous annexin I and the 10-kDa protein associated in a reversible Ca(2+)-dependent manner. Experiments with other annexins and with N-terminal truncated forms of annexin I indicated that the 10-kDa protein bound specifically to a site within the first 12 amino acids of annexin I. The 10-kDa protein was purified from human placenta by hydrophobic and affinity chromatography. Amino acid sequence analysis indicated that the 10-kDa protein is the human homologue of S100C, a recently identified member of the S100 subfamily of EF-hand Ca(2+)-binding proteins.

The annexins are a structurally related family of Ca2+ and phospholipid binding proteins whose function has not been clearly defined. Further investigations of annexin function may be enhanced by studying simpler organisms that express fewer annexin gene products. We previously characterized annexin XII from the freshwater cnidarian Hydra vulgaris (Schlaepfer, D. D., D. A. Fisher, M. E. Brandt, H. R. Bode, J. Jones, and H. T. Haigler. 1992. J. Biol. Chem. 267:9529-9539). In this report, we detected one other hydra annexin (40 kD) by screening hydra cell extracts with antibodies raised against peptides from highly conserved regions of known annexins. The 40-kD protein was expressed at less than 1% of annexin XII levels. These biochemical studies indicate that hydra contain a very limited number of annexin gene products. The cellular hydra annexin distribution was analyzed by indirect immunofluorescence. Using affinity-purified antibodies to annexin XII, the epithelial battery cells were stained throughout the tentacle. A lower level of annexin XII staining was detected in peduncle region epithelial cells. No other cell types showed detectable annexin XII staining. The anti-peptide antibody that specifically detected the 40-kD hydra annexin, maximally stained the cytoplasm of nematocytes. The immunofluorescent results showed that annexin XII and the 40-kD annexin were not co-expressed in the same cells. Since the hydra annexins localized to specific subsets of the total hydra cell types, it is likely that these proteins perform specialized biological roles, and not general "housekeeping" functions which are part of the essential molecular machinery of all cells.

As a first step toward the elucidation of a simple animal model in which to investigate annexin function, we identified, isolated, and characterized a novel annexin from Hydra vulgaris, annexin XII. A hydra cDNA library was screened using a probe generated by polymerase chain reaction from primers based on the partial amino acid sequence of annexin XII. Annexin XII cDNA was cloned and the functional protein was expressed in high yields in Escherichia coli. The annexin XII cDNA sequence predicted a 316-amino acid protein that had between 44 and 54% sequence identity with the Ca2+-binding core domains of previously characterized vertebrate and Drosophila annexins. The amino-terminal domain of annexin XII did not have sequence similarity with other known annexins except at and around a site that resembled known protein kinase C (PKC) phosphorylation sites in other annexins. As anticipated from its sequence, annexin XII was a high affinity substrate for purified rat brain PKC; half-maximal phosphorylation occurred below 0.1 microM annexin XII, and incorporation of up to 0.8 mol of phosphate/mol of annexin XII was observed. A PKC-like activity in hydra extracts also phosphorylated annexin XII. In summary, hydra promises to be a valuable model system for investigating the biological function of annexins and for determining how this function is modulated by PKC phosphorylation.

Annexins are primarily intracellular proteins as would be predicted from their lack of hydrophobic signal sequences. However, we now report that the human prostate gland selectively secretes high concentrations of annexin 1 (also called lipocortin 1 and p35) and a proteolytic cleavage product, des1-29-annexin 1, into seminal plasma. Secreted annexin 1 had a blocked amino terminus and was structurally indistinguishable from intracellular annexin 1. Although annexin 1 and the structurally related protein, annexin 4, co-localized to many of the same cells of the ductal epithelium of the prostate, annexin 4 was not secreted. Thus, the secretion of annexin 1 appears to involve a highly selective mechanism that does not involve targeting to the endoplasmic reticulum by a hydrophobic signal sequence.

Annexins are a structurally related family of Ca2+ binding proteins of undertermined biological function. Annexin I (also called lipocortin 1) is a substrate for the EGF-stimulated tyrosine kinase and is postulated to be involved in mitogenic signal transduction. To investigate further the involvement of lipocortin 1 in cell proliferation, we measured lipocortin 1 levels in normal diploid human foreskin fibroblasts (HFF) to determine whether its expression changed as a function of growth status. For comparison, the expression of annexin V (also called endonexin II) was measured in HFF cells. Endonexin II is a protein with similar Ca2+ and phospholipid binding properties as lipocortin 1, but it is not a substrate for tyrosine kinases. Quiescent HFF cell cultures were induced to proliferate by either subculture to lower cell density, EGF stimulation, or serum stimulation. In all three protocols, proliferating HFF cells contained three- to fourfold higher levels of lipocortin 1 and three- to fourfold lower levels of endonexin II than quiescent HFF cells. In contrast, the expression of annexin II (also called calpactin I) and annexin IV (also called endonexin I) remained relatively unchanged in growing and quiescent HFF cells. Lipocortin 1 synthesis rate was eightfold higher and its turnover rate was 1.5-fold slower in proliferating compared to quiescent HFF cells. Endonexin II synthesis rate remained constant but its turnover rate was 2.2-fold faster in proliferating compared to quiescent HFF cells. In a separate set of experiments, annexin expression levels were measured in cultures of rat PC-12 cells, a pheochromocytoma that ceases proliferation and undergoes reversible differentiation into nondividing neuronlike cells in response to nerve growth factor (NGF). After NGF treatment, PC-12 cells expressed fivefold higher levels of endonexin II and 32-fold higher levels of calpactin 1. Lipocortin 1 and endonexin I were not expressed in PC-12 cells. In summary, lipocortin 1 expression exhibited a positive correlation with cell proliferation in HFF cells. The increased expression of endonexin II in quiescent HFF cells and differentiating PC-12 cells implies that this protein may play a more prominent role in nondividing cells.

Annexin V is a protein of unknown biological function that undergoes Ca(2+)-dependent binding to phospholipids located on the cytosolic face of the plasma membrane. Preliminary results presented herein suggest that a biological function of annexin V is the inhibition of protein kinase C (PKC). In vitro assays showed that annexin V was a specific high-affinity inhibitor of PKC-mediated phosphorylation of annexin I and myosin light chain kinase substrates, with half-maximal inhibition occurring at approximately 0.4 microM. Annexin V did not inhibit epidermal growth factor receptor/kinase phosphorylation of annexin I or cAMP-dependent protein kinase phosphorylation of the Kemptide peptide substrate. Since annexin V purified from both human placenta and recombinant bacteria inhibited protein kinase C activity, it is not likely that the inhibitor activity was associated with a minor contaminant of the preparations. The following results indicated that the mechanism of inhibition did not involve annexin V sequestration of phospholipid that was required for protein kinase C activation: similar inhibition curves were observed as phospholipid concentration was varied from 0 to 800 micrograms/mL; the extent of inhibition was not significantly affected by the order of addition of phospholipid, substrate, or PKC, and the core domain of annexin I was not a high-affinity inhibitor of PKC even though it had similar Ca2+ and phospholipid binding properties as annexin V. These data indirectly indicate that inhibition occurred by direct interaction between annexin V and PKC. Since the concentration of annexin V in many cell types exceeds the amounts required to achieve PKC inhibition in vitro, it is possible that annexin V inhibits PKC in a biologically significant manner in intact cells.

Human placental lipocortin is a high-affinity substrate for rat brain protein kinase C in vitro with phosphorylation occurring on serine and threonine residues in a ratio of approximately 2 to 1. Comparison of the ability of various N-terminal-truncated derivatives of lipocortin to serve as phosphorylation substrates, and direct analysis of the N-terminal peptides cleaved from /sup 32/P-labeled lipocortin, indicated that threonine-24, serine-27, and serine-28 were the phosphorylation sites. The possibility is discussed that a lysine residue near the carboxy side of the phosphorylation site was involved in lipocortin interaction with the catalytic site of protein kinase C.

Horseradish peroxidase (HRP) uptake was used to measure fluid-phase pinocytosis in monolayers of human epithelioid carcinoma cells (A-431). Histochemistry confirmed that cell-associated HRP was restricted to intracellular vesicles. Biochemical methods showed that HRP uptake in control cultures was directly proportional to the duration of exposure. The addition of low concentrations of epidermal growth factor (EGF) to the incubation media produced a 10-fold increase in the initial rate of pinocytosis. The EGF effect was rapid (within 30 s) but transient; the rate of pinocytosis returned to control levels within 15 min. Metabolic inhibitors reduced the EGF-stimulated rate of pinocytosis by greater than 90%. A conjugate of EGF and ferritin (F:EGF) was used to simultaneously compare the intracellular locations of EGF and HRP. Much of F:EGF was internalized in approximately 100-nm vesicles, while most of the HRP was located in much larger vesicles (range 0.1--1.2 micrometer) which also contained F:EGF. The tumor-promoter 12-0-tetradecanoyl-phorbol-13-acetate, which shares several biological activities with EGF, was also effective in stimulating an increase in the rate of pinocytosis.

Previous studies using a biologically active 1:1 conjugate of EGF and ferritin (F-EGF) have traced the binding and internalization of the hormone molecules. In the present report, we develop ultrastructural criteria for identification of the F-EGF·receptor complex, and, thereby, enable utilization of the F-EGF as an indirect marker to localize the receptor for this peptide hormone. The ferritin cores of bound F-EGF are situated 4-6 nm from the extracellular surface of the membrane. When cells were incubated for up to 30 min at 37°C, this characteristic spatial relationship was observed in all uptake stages (surface clustering, endocytosis, and incorporation into multivesicular bodies), indicating that the hormone·receptor complex remains intact through these steps. However, when incubation was continued for periods sufficient to allow hormone degradation (30-60 min), pools of free ferritin were observed in lysosomes. In the presence of various amine inhibitors of hormone degradation, internalization and multivesicular body incorporation proceeded, but hormone·receptor degradation was blocked as evidenced by preservation of the ferritin—membrane relationship; i.e., no pools of free ferritin were seen after 60 min. These data provide morphological support for the hypothesis that down-regulation of surface receptors involves internalization of intact hormone·receptor complexes. In addition, we have developed a method for viewing the surface of intact cells en face , allowing closer scrutiny of the clustering of F-EGF·receptor complexes in the plane of the membrane prior to internalization. The particles in the F-EGF clusters observed by this method are spaced at 12 nm center-to-center, serving to set upper limits on the packing dimensions of the EGF·receptor complex.

Lipocortin I is a high affinity substrate for the epidermal growth factor receptor/kinase that can be purified by reversible Ca2+-dependent association with cellular particulate fractions. Purified human lipocortin I was shown by ultraviolet spectroscopy to undergo conformational changes in response to high concentrations of Ca2+ (30-700 microM). Equilibrium dialysis of lipocortin I alone against 45Ca2+ showed barely detectable binding at 5-60 microM Ca2+. However, in the presence of phosphatidylserine, binding was significantly enhanced, and Scatchard analysis of the binding data showed that lipocortin I contained four Ca2+-binding sites with an apparent Kd of 75 microM. Lipocortin I associated with phosphatidylserine vesicles and F-actin in a Ca2+-dependent manner with half-maximal association occurring at 22 and 180 microM Ca2+, respectively. Neither phosphatidylcholine vesicles nor intact cultured fibroblasts bound detectable amounts of lipocortin I. Phosphorylation of lipocortin I by the epidermal growth factor-stimulated kinase in A431 membranes required Ca2+ (half-maximum = 28 microM) in the presence of Mg2+, but phosphorylation was not Ca2+-dependent in the presence of Mn2+. The difference in Ca2+ requirement for phosphorylation probably is a reflection of the fact that Mn2+, but not Mg2+, promoted the association of lipocortin I with phospholipid. Although the physiological role of lipocortin I phosphorylation has not yet been determined, it was observed that phosphorylation of Tyr-21 reduced by 5-fold the amount of Ca2+ required for half-maximal association of the protein with phosphatidylserine vesicles.

We have prepared several electron and light microscopic labels of epidermal growth factor (EGF) to analyse the morphologic features of its binding and internalization by cultured cells. These include a ferritin conjugate of EGF, a covalent conjugate of EGF and horseradish peroxidase (EGF-HRP), a colloidal gold marker system using EGF-HRP as a primary antigen, and a covalent complex of EGF with rhodamine-labelled lactalbumin. All of the light and electron microscopic labels showed similar patterns of binding. EGF initially bound to diffusely distributed cell surface receptors at 4 degrees C. The EGF-receptor complexes clustered into clathrin-coated pits on the cell surface only when the temperature was raised to 37 degrees C. In KB and Swiss 3T3 cells, this was followed by rapid internationalization into receptosomes, compartmentalization into the Golgi system, clustering in the clathrin-coated regions of the Golgi, and finally delivery into lysosomes from the Golgi. This general pathway was seen in Swiss 3T3 cells which have a low number of EGF receptors, KB cells which have a moderate number of receptors and A431 cells that have a high number of receptors. However, the ruffling activity induced in A431 cells by EGF produced some internalization through macropinosomes, making the pathway of entry more difficult to evaluate. Double label experiments showed that EGF is internalized together with alpha 2-macroglobulin and adenovirus particles. These data clarify the route of entry of EGF in different cell types using multiple labels, and shows that it enters cells through the same coated pit entry pathway as most other ligands previously examined.

Endonexin II is a member of the family of Ca2+-dependent phospholipid binding proteins known as annexins. We cloned human endonexin II cDNA and expressed it in Escherichia coli. The apparent size and Ca2+-dependent phospholipid binding properties of purified recombinant endonexin II were indistinguishable from those of the placental protein. A single mRNA of approximately 1.6 kilobase pairs was found to be expressed in human cell lines and placenta and was in close agreement with the length of the cDNA clone (1.59 kilobase pairs). The cDNA predicted a 320-amino acid protein with a sequence that was in agreement with the previously determined partial amino acid sequence of endonexin II isolated from placenta. Endonexin II contained 58, 46, and 43% sequence identity to protein II, calpactin I (p36, protein I), and lipocortin I (p35), respectively. The partial sequence of bovine endonexin I was aligned with the sequence of endonexin II to give 63% sequence identity. Like these other proteins, endonexin II had a 4-fold internal repeat of approximately 70 residues preceded by an amino-terminal domain lacking similarity to the repeated region. It also had significant sequence identity with 67-kDa calelectrin (p68), a protein with an 8-fold internal repeat. Comparing the amino-terminal domains of these four proteins of known sequence revealed that, in general, only endonexin II and protein II had significant sequence identity (29%). Endonexin II was not phosphorylated by Ca2+/phospholipid-dependent enzyme (protein kinase C) even though it contained a threonine at a position analogous to the protein kinase C phosphorylation sites of lipocortin I, calpactin I, and protein II.

We have investigated the epidermal growth factor (EGF)-stimulated tyrosine-specific protein kinase activity in quiescent cultures of diploid human fibroblasts that have a well characterized mitogenic response to EGF. We developed a method of permeabilizing cells with digitonin or other agents that permitted the rapid labeling of cellular proteins with exogenously added [gamma-32P]ATP while allowing only about 25% of marker cytosolic enzymes to escape from the cells. When phosphatases were inhibited with zinc and vanadate, EGF induced up to 8-fold stimulation of the incorporation of radioactivity from [gamma-32P]ATP into a 35-kDa band on sodium dodecyl sulfate gels. Alkali treatment of gels showed that EGF stimulated the phosphorylation of bands with apparent molecular masses of 170, 45, 35, 26, 22, and 21 kDa. Phosphoamino acid analysis was performed on the 170- and 35-kDa bands and revealed that the EGF-stimulated phosphorylation was on tyrosyl residues. The 35-kDa band was resolved into four spots by two-dimensional gel electrophoresis. The most acidic form was the most prominent and it was precipitated by an antiserum against a 35-kDa protein from A-431 cells; heretofore, this protein has only been reported to be phosphorylated in an EGF-dependent manner by A-431 membranes in vitro (Fava, R. A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). This antiserum also precipitated a 35-kDa phospho-protein from extracts of intact [32P]orthophosphate-labeled fibroblasts which was phosphorylated on tyrosine in an EGF-dependent manner. None of the forms of the 35-kDa phosphoproteins labeled in permeabilized cells were immunologically related to the 34-kDa protein that is a substrate for the tyrosyl kinase encoded by Rous sarcoma virus. Other mitogens (serum, insulin, platelet-derived growth factor, and thrombin) did not detectably stimulate phosphorylation in permeabilized cells.

Reversible calcium-dependent association with a particulate fraction from human placenta was used as the first step in the purification of substrates for the epidermal growth factor-stimulated protein kinase. A protein with apparent Mr of 35,000 was purified to homogeneity, and the sequence was determined for approximately one-fourth of the protein. These residues could be aligned exactly with the previously published sequence of lipocortin I derived from the cDNA from a human lymphoma. Two other proteins that appear to be formed by proteolytic removal of 12 or 26 of the amino acids from the NH2 terminus of the protein also were isolated. Placental lipocortin I was phosphorylated in Tyr-21 in an epidermal growth factor-dependent manner by the kinase activity in a particulate fraction from A431 cells; half-maximal phosphorylation occurred at 50 nM lipocortin I. Lipocortin I phosphorylated on Tyr-21 was approximately 10-fold more sensitive to tryptic cleavage at Lys-26 than was the native protein. Placental lipocortin I and its two truncated forms were potent inhibitors of pancreatic phospholipase A2 activity. Another 33-kDa protein that was not related immunologically to lipocortin I or lipocortin II (calpactin I) also was purified from the EGTA extract of placenta. The unidentified protein inhibited phospholipase A2 but was not a substrate for the epidermal growth factor-stimulated kinase. The mechanism by which these proteins inhibit phospholipase A2 activity was investigated. Attempts to detect direct interaction between these proteins and the enzyme were unsuccessful. However, both the unidentified protein, lipocortin I, and 32P-labeled lipocortin I bound in a Ca2+-dependent manner to the [3H]oleic acid-labeled Escherichia coli membranes used as substrate in the phospholipase A2 assay. Heparin, which is known to block lipocortin I inhibition of phospholipase A2, also blocked binding of lipocortin I to E. coli membranes. The results of these and other experiments raise the possibility that placental lipocortin I inhibits phospholipase A2 activity in this assay by coating the phospholipid and thereby blocking interaction of enzyme and substrate.

Radiation inactivation with high energy electrons from a linear accelerator was used to determine the functional molecular size of the epidermal growth factor (EGF) binding site and the tyrosine-specific protein kinase activity in A-431 membranes. The target size of the protein portion of the EGF receptor glycoprotein was 147,000 daltons when the radiation-dependent decrease in maximal binding capacity was measured. Since the target size is in good agreement with the molecular size of the protein portion of the EGF receptor determined by denaturing biochemical methods, it appears that the monomeric receptor is the functional binding site in situ. The target size of the EGF-stimulated kinase activity associated with the affinity-purified EGF receptor/kinase was 133,000 and 144,000 daltons when assayed for the ability to autophosphorylate or to phosphorylate a tyrosine-containing peptide, respectively. However, the target size of the kinase activity that did not adhere to an EGF-affinity column was 54,000 and 69,000 daltons when assayed for phosphorylation of endogenous and exogenous substrates, respectively. Intermediate target sizes were obtained when kinase assays were performed on membranes prior to fractionation by affinity chromatography. These results, taken with other biochemical data, indicate that A-431 membranes contain a kinase activity that is a domain of the glycoprotein that contains the EGF binding site and that the membranes also contain another tyrosine-specific kinase or kinases that have an average size of approximately 60,000 daltons.

The ability of rat liver, kidney, muscle, heart and testis tissue to carry out the in vitro synthesis of carnitine via e-N-trimethyllysine and γ-butyrobetaine was studied. All tissues formed γ-butyrobetaine from trimethyllysine, but liver and testis also formed carnitine in about 7% and 1% yield respectively. Liver slices formed trimethyllysine from lysine in about 6% yield. These in vitro studies thus establish that liver has all the enzymes of the carnitine biosynthetic pathway. This tissue appears to be the primary site of carnitine synthesis in the rat as implied from whole animal studies in this and other laboratories.

Annexin 5 is a Ca 2+ -binding protein, the function of which is poorly understood. Structural and electrophysiological studies have shown that annexin 5 can mediate Ca 2+ fluxes across phospholipid membranes in vitro [1]. There is, however, no direct evidence for the existence of annexin 5 Ca 2+ channels in living cells. Here, we show that annexin 5 inserts into phospholipid vesicle membranes at neutral pH in the presence of peroxide. We then used targeted gene disruption to explore the role of annexin 5 in peroxide-induced Ca 2+ signaling in DT40 pre-B cells. DT40 clones lacking annexin 5 exhibited normal Ca 2+ responses to both thapsigargin and B-cell receptor stimulation, but lacked the sustained phase of the response to peroxide. This late phase was due to Ca 2+ influx from the extracellular space, demonstrating that annexin 5 mediates a peroxide-induced Ca 2+ influx. Thus, peroxide induces annexin 5 membrane insertion in vitro , and peroxide-induced Ca 2+ entry in vivo in DT40 cells requires annexin 5. Our results are consistent with a role for annexin 5 either as a Ca 2+ channel, or as a signaling intermediate in the peroxide-induced Ca 2+ -influx pathway.

The binding and internalization of 125I-epidermal growth factor (125I-EGF) was studied in cultures of BALB 3T3 cells using a novel method that involved removal of cell-surface hormone by treatment with acetic acid under conditions that did not remove internalized hormone. In control cultures, 125I-EGF initially bound to its receptor on the plasma membrane and then was rapidly internalized. After 30 min, only 15% of the cell-bound hormone remained on the surface. In contrast, cultures treated with dansylcadaverine retained 82% of the cell-bound hormone on the cell surface. We propose that dansylcadaverine inhibits EGF internalization by preventing it from clustering in clathrin-coated pits.

The annexins are a family of phospholipid- and Ca2+-binding proteins that are structurally related. Two members of this family, human endonexin II and chicken anchorin CII, may arise from the same gene by alternative splicing of two structurally unrelated segments.

Publisher Summary This chapter describes purification of synexin and methods for analyzing synexin activity. To measure synexin activity, a recording spectrophotometer such as the Gilson 250 can be used, with which four samples can be measured at one time. In addition, a four-cuvet holder is needed, as are the cuvets. The cuvets can either be quartz or acrylic plastic, and of a size suitable for holding 1 ml total volume. The recorder should be set with a full scale of OD 0.2 at 540 nm. The instrument can be temperature statted with a circulating water pump, but routine assays are usually performed at room temperature. The chapter also describes techniques for purifying and assaying endonexin II and lipocortin I.

The lateral mobility of the epidermal growth factor (EGF) receptor in the plane of the plasma membrane of cultured A431 cells was investigated using direct and indirect fluorescent probes to measure the generation and relaxation of electric field-induced receptor asymmetry. A steady electric field of 15 V/cm for 30 min at 23 degrees C induced a redistribution of the unoccupied EGF receptor such that there was approximately a three-fold higher concentration of receptors at the cathode-facing pole. After termination of the field, the unoccupied receptors back diffused at 37 degrees C with a rate corresponding to a diffusion coefficient of 2.6-3.5 X 10(-10) cm2/s. No diffusion was detected at 4 degrees C. Formation of the hormone-receptor complex is known to induce receptor clustering and internalization. By inhibiting internalization with metabolic poisons, we were able to study the cell surface mobility of clusters of the hormone-receptor complex. The same degree of asymmetry was induced when the occupied receptor was exposed to an electric field and the rate of back diffusion of clusters of the hormone-receptor complex corresponded to a diffusion coefficient of 0.68-0.95 X 10(-10) cm2/s. Although the unoccupied receptor is somewhat more mobile than the hormone-receptor complex, it was still far less mobile than one would predict for an unconstrained protein imbedded in a phospholipid bilayer.

To examine in vivo phosphorylation of lipocortin I we made use of a polyclonal antibody to an amino terminal peptide of lipocortin I. This antibody does not recognize any other member of the annexin protein family, and can both immunoprecipitate lipocortin I and recognize this protein on western blots. Using cleaved forms of lipocortin I, we have been able to demonstrate that protein kinase C phosphorylates this protein in vitro within the first 29 amino terminal amino acids. However, the addition of phorbol esters to A431 cells over a wide range of concentrations and for varying periods of time did not stimulate the phosphorylation of this protein. Since in vitro lipocortin I is an excellent substrate for all three isoforms, alpha, beta, gamma, of protein kinase C, the discrepancy in these findings is not secondary to the presence of varying forms of this protein kinase within different cell types.

Membranes were prepared from the human epithelioid carcinoma cell line A-431 which has approx. 2 . 10(6) epidermal growth factor receptors per cell. This membrane preparation which retained a high epidermal growth factor binding specific activity was used as an antigen to produce antisera in rabbits. Double-immunodiffusion experiments demonstrated that the immune serum contained precipitating antibodies to several components of detergent solubilized A-431 membranes. The immunoglobulin G fraction of this immune sera inhibited 125I-labeled epidermal growth factor binding to receptors in: (1) intact human and mouse cells; (2) membrane preparations from A-431 cells and human placenta, and (3) solubilized A-431 membranes. Inhibition of 125I-labeled epidermal growth factor binding was observed with divalent and monovalent fragments of immunoglobulin G prepared from the immunoglobulin G fraction. Also, the immunoglobulin G fraction blocked growth factor binding to membranes at low temperature (5 degrees C). Anti-A-431 antibody blocked the induction of DNA synthesis in quiescent fibroblasts by epidermal growth factor in a manner similar to that of anti-epidermal growth factor antibody. Addition of either anti-A431 or anti-epidermal growth factor antibodies to fibroblasts at times up to 5 h after the addition of epidermal growth factor completely reversed the hormone's mitogenic potential. At later times (after 12 h) addition of either antibody was without effect on the stimulation of DNA synthesis by epidermal growth factor. Anti-A-431 antibody did not block the induction of DNA synthesis in fibroblasts by fibroblast growth factor or serum.

Recent studies have defined a family of structurally related proteins which bind to certain phospholipids in Ca2+-dependent manner (Crompton et al., 1988). Several names have been proposed for this family including annexins, lipocortins, and calpactins. Although the biological function is not known for any of these proteins, they are attracting intensive investigation because of their potential involvement in Ca2+-mediated stimulus-response coupling.

The binding and internalization of epidermal growth factor (EGF) in human epithelioid carcinoma cells (A-431), which have approximately 2.6 X 10(6) receptors per cell, has been followed with 125I-labeled EGF and by fluorescence microscopy. We have prepared a fluorescent derivative of EGF that is biologically active and retains substantial binding affinity for cell receptors. After binding of this derivative to cells at 6 degrees, the cellular borders were prominently stained and the fluorescence on the remainder of the membrane was uniform. Upon warming of these cells to 37 degrees for 10 min, the surface fluorescence diminished and randomly distributed endocytotic vesicles appeared in the cytoplasm. After 20 min at 37 degrees these fluorescent vesicles formed a perinuclear ring. The binding of EGF to the surface of these cells was also visualized by immunofluorescence using rabbit antibodies to EGF and rhodamine-labeled goat anti-rabbit antibodies. We did not detect large fluorescent clusters or cap formation in these experiments. These data provide direct confirmation of the previous biochemical data that suggested that cell membrane-bound EGF is rapidly internalized.

We have investigated the cellular fate of epidermal growth factor (EGF) in KB cells and a variant, KB-R2A, that was isolated based on its resistance to diphtheria toxin and subsequently was shown to be resistant to infection by RNA viruses (Moehring and Moehring, 1972, Infect. Immunity. 6:487-492). Both cell lines bind 125I-EGF and internalize the cell-bound hormone at the same rate. However, when the degradation of internalized 125I-EGF was measured by the release of low molecular weight (mw) hydrolysis products into the medium, the toxin-resistant KB-R2A cells degraded the hormone at a drastically reduced rate; 50% and 3% of the cell-bound 125I-EGF was degraded and released by 80 min in the KB and KB-R2A cells, respectively. To investigate the fate of cell-associated EGF prior to release into the medium, the radioactivity in extracts of cells labeled with 125I-EGF was fractionated by native gel electrophoresis. In KB cells three peaks of radioactivity other than native 125I-EGF were resolved. Time course and subcellular fractionation studies showed that the first processed product appeared while the hormone was located in the endocytic vesicles and the appearance of the other two peaks correlated with the arrival of the hormone in the lysosomal compartment. KB-R2A cells also produced the first intermediate but they produced only very low amounts of the other two peaks. These studies show that endocytic vesicles in both cell lines contain enzymes capable of processing EGF prior to the arrival of the hormone in the lysosomes and show that the KB-R2A cells have a lesion that prevents the complete degradation of the hormone. We propose that the KB-R2A cell line has a defective mechanism for the intracellular processing of a number of ligands that are internalized by the process of receptor-mediated endocytosis and that this defect is located beyond the initial endocytic step.

Publisher Summary This chapter focuses on the receptor-mediated endocytosis of epidermal growth factor (EGF). The EGF is a stable, well-characterized polypeptide that can be isolated in relatively large amounts from the mouse submaxillary gland and, therefore, lends itself to chemical modification. Radioactive, fluorescent, and ferritin conjugates of EGF can be prepared that bind to cellular receptors in a highly specific manner and permit the study of the interactions of EGF with its target cells at different levels of resolution. With these derivatives, it has been shown that EGF binds to specific receptors on the plasma membrane that are initially diffusely distributed. The hormone-receptor complex then clusters in clathrin-coated pits and is internalized into endocytic vesicles that eventually deliver their contents to lysosomes. Fluorescein-conjugated EGF (F1-EGF) can be prepared by allowing EGF to react with fluorescein isothiocyanate (FITC). The chapter discusses a method that permits the study of EGF internalization using a simple biochemical method that allows a clear discrimination between cell-surface-bound and internalized hormone.