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Biomedical subjects

L A Greene

Publications and source records attributed to L A Greene.

At least 163 records · Page 9Linked to original sources
PubMed1985

Cell surface Thy-1-cross-reactive glycoprotein in cultured PC12 cells: modulation by nerve growth factor and association with the cytoskeleton.

In cultured PC12 rat pheochromocytoma cells, nerve growth factor (NGF) selectively stimulated the incorporation of [3H]fucose into a glycoprotein of apparent Mr approximately 25,000, as determined by sodium dodecyl sulfate-gel electrophoresis. Neither dibutyryl cyclic adenosine 3':5'-monophosphate nor epidermal growth factor mimicked this effect. Using gradient gels, the affected protein was resolved into two closely migrating bands. Enhancement of labeling was present by 1 to 2 days of treatment with NGF and was maximal after 4 to 7 days. Short-term extraction of PC12 cell monolayer cultures with Triton X-100 left the 25,000-dalton glycoprotein associated with the detergent-resistant cytoskeletal fraction. The Mr approximately 25,000 glycoprotein was shown to be immunologically cross-reactive with Thy-1.1 antigen by indirect immunoprecipitation with monoclonal Thy-1.1 antibodies. Anti-Thy-1.1 labeling, as demonstrated by indirect immunofluorescence, was distributed on PC12 cell bodies and along neuritic processes and remained attached to the cytoskeleton as part of the surface lamina of cells treated and untreated with NGF. The selective increase of the Mr approximately equal to 25,000 glycoprotein in NGF-treated cultures was also paralleled by increases in material immunoprecipitated from fucose-labeled cells with anti Thy-1.1 monoclonal antibodies. Immunoprecipitations with extracts of [35S]methionine-labeled cultures indicated that NGF causes an increase in synthesis, rather than merely glycosylation, of the 25,000-dalton/Thy-1-cross-reactive protein. The effect of NGF on this protein was blocked by inhibitors of RNA synthesis, suggesting involvement of a transcription-dependent mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Regulation of tyrosine hydroxylase phosphorylation in PC12 pheochromocytoma cells by elevated K+ and nerve growth factor. Evidence for different mechanisms of action.

A specific antiserum was used to compare phosphorylation of tyrosine hydroxylase (TH) (EC 1.14.16.2, tyrosine 3-monooxygenase) as regulated by elevated K+ and nerve growth factor (NGF) in cultured PC12 pheochromocytoma cells. Exposure of cultures to either elevated K+ or to NGF significantly enhanced the incorporation of [32P]orthophosphate into TH. The effect of elevated K+ was evident at 10 mM and was maximal by 40-80 mM. Increased phosphorylation of TH was detected at 0.1 nM (3 ng/ml) NGF and reached a maximal level by 0.3-1 nM (10-30 ng/ml) NGF. Elevated K+ showed a biphasic time course of action with one maximum of phosphorylation at about 30 sec of exposure and a second after about 10 min of exposure. In contrast, the NGF effect showed an initial lag of several minutes followed by a monophasic increase in phosphorylation to reach a plateau. Both treatments enhanced TH activity, but in each case the time courses of this did not strictly correlate with that of phosphorylation. The effect of elevated K+ on TH phosphorylation required the presence of extracellular Ca2+ and was suppressed by trifluoperazine (100 microM). N-(6-Aminohexyl)-5-(chloronaphthalene)-1-sulfonamide (W-7) (100 microM), a potent inhibitor of calmodulin activity, also blocked the enhancement of phosphorylation by elevated K+, whereas N-(6-aminohexyl)-1-(naphthalene)sulfonamide (W-5) (100 microM), a less potent analogue of W-7, did not. In contrast to these findings, the increase in TH phosphorylation brought about by NGF did not require extracellular Ca2+, and was only slightly affected by trifluoperazine or W-7. When TH phosphorylated under various conditions (control medium, elevated K+, NGF) was subjected to peptide mapping after exposure to Staphylococcus aureus protease V8, multiple phosphorylated peptides were observed. Elevated K+ and NGF each produced increases in labeling of each of the peptides. However, the relative degree of labeling of different peptides was distinct for each condition. These data suggest that elevated K+ and NGF bring about rapid enhancement of the phosphorylation of TH by means of different mechanisms.

Animals↗
PubMed1984

Association of 125I-nerve growth factor with PC12 pheochromocytoma cells. Evidence for internalization via high-affinity receptors only and for long-term regulation by nerve growth factor of both high- and low-affinity receptors.

Association of 125I-nerve growth factor (NGF) with PC12 pheochromocytoma cells was studied. Surface-bound and internalized NGF were distinguished by differential release of the former at low pH, high salt. Binding to the surface was rapid; at 0.2 nM (5 ng/ml) 125I-NGF, this was near-maximal within 5 min. Internalization, in contrast, did not start until about 2 min after NGF exposure and, thereafter, proceeded linearly for at least 1/2-1 h. By the latter time, approximately 75% of total bound NGF was within rather than on the surface of the cells. Binding versus concentration experiments indicated two distinct classes of surface binding sites. For both naive cells and cells treated with NGF for at least a week (primed cells), about 7% of the receptors had an apparent binding constant of about 0.3 nM; the remaining sites half-saturated at approximately 4 nM NGF. The number of each type of site was 3--4-fold higher/mg of protein in primed cells. For both naive and primed cultures, internalization appeared to be mediated by a single class of uptake sites which half-saturated at about 0.3 nM. The maximal rate of uptake by primed cells (200 fmol/h/mg protein) was about twice that for naive cells. Light and electron microscopic autoradiography indicated that the density of binding was substantially higher in primed cultures and that this increase took place over a time course of days to weeks. These findings suggest that NGF brings about long-term increases in its own high- and low-affinity surface receptors, but is internalized only via the high-affinity sites.

Adrenal Gland Neoplasms↗
PubMed1984

Fucosyl gangliosides of PC12 pheochromocytoma cells.

Three monosialogangliosides are highly labeled when PC12 pheochromocytoma cells are cultured in the presence of L-[3H]fucose, and two additional monosialogangliosides are labeled to a lesser extent. In contrast, neither of the two disialogangliosides of PC12 cells contains fucose residues. Removal of sialic acid and fucose by formic acid hydrolysis demonstrated the presence of 3 major 'core' structures in the monosialogangliosides, and a single asialo derivative of the disialogangliosides which has the same chromatographic mobility as one of the monosialoganglioside hydrolysis products. None of the major formic acid hydrolysis products of the PC12 cell gangliosides corresponds to asialo-GM1, supporting our previous conclusion that PC12 cells do not contain significant amounts of brain-type gangliosides.

Adrenal Gland Neoplasms↗
PubMed1984

Calcium/phospholipid-dependent protein kinase (protein kinase C) phosphorylates and activates tyrosine hydroxylase.

Protein kinase C, purified to homogeneity, was found to phosphorylate and activate tyrosine hydroxylase that had been partially purified from pheochromocytoma PC 12 cells. These actions of protein kinase C required the presence of calcium and phospholipid. This phosphorylation of tyrosine hydroxylase reduced the Km for the cofactor 6-methyltetrahydropterine from 0.45 mM to 0.11 mM, increased the Ki for dopamine from 4.2 microM to 47.5 microM, and produced no change in the Km for tyrosine. Little or no change in apparent Vmax was observed. These kinetic changes are similar to those seen upon activation of tyrosine hydroxylase by cAMP-dependent protein kinase. Two-dimensional phosphopeptide maps of tyrosine hydroxylase were identical whether the phosphorylation was catalyzed by protein kinase C or by the catalytic subunit of cAMP-dependent protein kinase. Both protein kinases phosphorylated serine residues. The results suggest that protein kinase C and cAMP-dependent protein kinase phosphorylate the same site(s) on tyrosine hydroxylase and activate tyrosine hydroxylase by the same mechanism.

Animals↗
PubMed1984

Differential inhibition of nerve growth factor and epidermal growth factor effects on the PC12 pheochromocytoma line.

Tests have been made of the action of the methyltransferase inhibitors 5'-S-methyl adenosine, 5'-S-(2-methyl-propyl)-adenosine, and 3-deaza-adenosine +/- L-homocysteine thiolactone, on nerve growth factor (NGF)-dependent events in the rat pheochromocytoma line PC12. Each of these agents inhibited NGF-dependent neurite outgrowth at concentrations of the order of millimolar. Slow initiation of neurite outgrowth over several days and more rapid regeneration of neurites (congruent to 1 d) were blocked, as was the priming mechanism necessary for genesis of neurites. The inhibitions were reversible in that PC12 cells maintained for several days in the presence of inhibitors grew neurites normally after washout of these agents. Other NGF-dependent responses of the PC12 line (i.e., induction of ornithine decarboxylase activity [over 4 h], enhancement of tyrosine hydroxylase phosphorylation [over 1 h], and rapid changes in cell surface morphology [30 s onward]) were inhibited by each of the agents. In contrast, corresponding epidermal growth factor-dependent responses in ornithine decarboxylase activity, phosphorylation, and cell surface morphology were not blocked, but instead either unaffected or enhanced, by the methylation inhibitors. These inhibitors did not act by blockade of binding of NGF to high- or low-affinity cell surface receptors, though they partially inhibited internalization of [125I]NGF. The inhibition of rapidly-induced NGF-dependent events and the differential inhibition of responses to NGF and epidermal growth factor imply that the methyltransferase inhibitors specifically block one of the first steps in the mechanistic pathway for NGF.

Adrenal Gland Neoplasms↗
PubMed1984

Comparison of rapid changes in surface morphology and coated pit formation of PC12 cells in response to nerve growth factor, epidermal growth factor, and dibutyryl cyclic AMP.

Scanning and transmission electron microscopic studies were carried out on the rapid cell surface response of PC12 pheochromocytoma cells to treatment with nerve growth factor (NGF), epidermal growth factor (EGF), and dibutyryl cyclic AMP. EGF induced a rapidly initiated series of surface changes identical to those previously observed with NGF. Ruffles appear over the dorsal surface of the cells by 30 s, are prominent at 3 min, and are absent by 7 min. Microvilli disappear as dorsal ruffles become prominent. Peripheral ruffles are seen by 3 min, are prominent on most of the cells by 7 min, and are virtually absent by 15 min. Large blebs are present on 50% of the cells by 2 h and are markedly decreased by 4 h. Within 30 s after NGF or EGF addition, an increase in the density of 60-130-nm coated pits per unit membrane is detectable. This reaches a maximum of two- to threefold in from 1 to 3 min and gradually decreases. Combined treatment with NGF and EGF increases surface ruffling and, after an early peak in coated pits which at 3 min is similar in magnitude to that observed for the separately administered factors, maintains a greater number of pits per unit area than either treatment alone. 3-d pretreatment with NGF greatly reduces the response of the cells to EGF both with respect to surface ruffling and coated pit formation while 4-h NGF pretreatment has no effect on the EGF response. Dibutyryl cyclic AMP induced none of the rapidly onsetting changes caused by NGF or EGF, and therefore it seems unlikely that cyclic AMP mediates these surface changes. Changes in cell surface architecture induced by NGF and EGF on PC12 cells and by NGF in normal sympathetic neurons (as previously described) indicates that such responses may be a widespread phenomenon associated with the interaction of at least some peptide growth factors/hormones with their receptors. These responses may represent or reflect primary events in the mechanism by which these factors act.

Adrenal Gland Neoplasms↗
PubMed1984

Rapid activation of tyrosine hydroxylase in response to nerve growth factor.

Nerve growth factor protein (NGF) was found to rapidly promote the activation of tyrosine hydroxylase in cultured rat PC12 pheochromocytoma cells. PC12 cultures were exposed to NGF for periods of less than 1 h and the soluble contents of homogenates prepared from the cells were assayed for tyrosine hydroxylase activity. Under these conditions, the specific enzymatic activity was increased by 60 +/- 10% (n = 13) in comparison with that in untreated sister cultures. The increase was half maximal by 2-5 min of exposure and at NGF concentrations of about 10 ng/ml (0.36 nM). Antiserum against NGF blocked the effect. Tyrosine hydroxylase activity could also be rapidly increased by NGF in cultures of PC12 cells that had been treated with the factor for several weeks in order to produce a neuron-like phenotype. This was achieved by withdrawing NGF for about 4 h and then readding it for 30 min. The NGF-induced increase of tyrosine hydroxylase activity in PC12 cultures was not affected by inhibition of protein synthesis and therefore appeared to be due to activation of the enzyme. Kinetic experiments revealed that NGF brought about no change in the apparent Km of the enzyme for tyrosine or for cofactor (6- methyltetrahydropteridine ), but that it did significantly increase the apparent maximum specific activity of the enzyme. These observations suggest that NGF (perhaps released by target organs) could promote a rapid and local enhancement of noradrenergic transmission in the sympathetic nervous system.

Adrenal Gland Neoplasms↗
PubMed1984

Release of the NILE and other glycoproteins from cultured PC12 rat pheochromocytoma cells and sympathetic neurons.

Studies were carried out on the glycoproteins (GPs) released by cultured rat sympathetic neurons and by cultured PC12 rat pheochromocytoma cells with and without nerve growth factor (NGF) treatment. Cultures were prelabeled with [3H]fucose and then incubated for 4-8 h in fresh unlabeled medium. The material released into the medium was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. The patterns of labeled material released by all three types of cultures were similar. One of the major components released was of apparent Mr less than or equal to 230,000. Another major component of apparent Mr = 55,000 as well as minor components of apparent Mr less than or equal to 180,000, 140,000, 118,000, and 105,000 were also detected. An additional peptide of apparent Mr less than or equal to 210,000 was released only by the sympathetic neurons. The soluble released Mr less than or equal to 230,000 component appeared to be derived from a previously characterized neuronal integral membrane GP referred to as the NILE (NGF-inducible large external) GP. Evidence for this included recognition of the released component by a monospecific antiserum prepared against membrane-derived NILE GP. At least several of the other released GPs appeared to be derived from membrane-bound components with which they share immuno-crossreactivity. Since the soluble NILE and other released GPs had somewhat faster mobilities on SDS-polyacrylamide gels than their apparent membrane-bound correspondents, release could either be due to, or accompanied by, minor changes in molecular structure.

Adrenal Gland Neoplasms↗
PubMed1983

Regulation of the multiple forms of dopamine beta-hydroxylase by nerve growth factor, dexamethasone, and dibutyryl cyclic AMP in the PC12 pheochromocytoma cell line.

Treatment with nerve growth factor was found to influence the subunit forms of dopamine beta-hydroxylase in PC12 pheochromocytoma cells. In untreated cells, near equal amounts of two subunit forms were observed (apparent Mr = 77,000 and 73,000) by labeling with [35S]methionine. Upon treatment of PC12 cells with nerve growth factor for several days, the Mr = 73,000 subunit form of dopamine beta-hydroxylase was almost exclusively observed. The shift in subunit forms became apparent only after a day of treatment and was maximal with 3 days or more of exposure to nerve growth factor. The dose-response curve was similar to most other nerve growth factor-induced responses in PC12 cells. Neurite outgrowth, however, was not essential for the shift in predominance of the Mr = 73,000 subunit form. This effect of nerve growth factor also occurred in suspension cultures or in the presence of low concentrations of inhibitors of transcription sufficient to prevent neurite outgrowth. Pulse-chase experiments with nerve growth factor-treated cells indicated that the Mr = 77,000 form is initially synthesized (5 min) and is then converted to the Mr = 73,000 form by 30-60 min. Insulin (100 ng/ml) and epidermal growth factor (1 ng/ml) had no effect on the subunit forms of dopamine beta-hydroxylase. However, treatment of PC12 cells for several days with dexamethasone (10(-5)M) or dibutyryl cyclic AMP (1 mM) leads to predominance of the Mr = 73,000 form of the enzyme. These experiments suggest that the proportions of the subunit forms of dopamine beta-hydroxylase can be regulated in cells by external signals and this may reflect alterations in post-translational processing enzymes and may serve as a potential mechanism to regulate catecholamine metabolism.

Adrenal Gland Neoplasms↗
PubMed1983

Mechanism of biosynthesis of soluble and membrane-bound forms of dopamine beta-hydroxylase in PC12 pheochromocytoma cells.

Dopamine beta-hydroxylase was present as 2 subunit forms (apparent Mr = 77,000 and 73,000) in the PC12 pheochromocytoma cell line as detected by immunoprecipitation from [35S]methionine-labeled cultures, and analyzed by sodium dodecyl sulfate gel electrophoresis and fluorography. The Mr = 77,000 form was present in a crude membrane fraction, while the Mr = 73,000 form was soluble. Both forms appeared to be present in approximately equal amounts, and both were glycosylated. Treatment of PC12 cells with tunicamycin, a potent inhibitor of core glycosylation in the endoplasmic reticulum, completely inhibited the appearance of the Mr = 77,000 and Mr = 73,000 forms, and 2 new immunoreactive polypeptides were obtained (apparent Mr = 67,000 and 63,000). Pulse-chase experiments suggested that the Mr = 77,000 form is initially synthesized (by 5 min) and a portion is converted in 15-90 min to the Mr = 73,000 form. Thereafter, the ratio between forms remains relatively constant, at least for several hours. Translation of mRNA from bovine and rat adrenals, and immunoprecipitation, indicated that dopamine beta-hydroxylase is initially synthesized as a single polypeptide (apparent Mr = 67,000). The subcellular site of biosynthesis of dopamine beta-hydroxylase was determined by isolation of mRNA from free and membrane-bound polysomes from bovine adrenal medulla. Translation in a cell free system and immunoprecipitation localized the synthesis of dopamine beta-hydroxylase on membrane-bound polysomes. These experiments suggest that both soluble and membrane-bound forms of dopamine beta-hydroxylase are synthesized and core glycosylated in the endoplasmic reticulum, and that there probably is a precursor-product relationship between the Mr = 77,000 and the Mr = 73,000 subunit forms of dopamine beta-hydroxylase.

Adrenal Gland Neoplasms↗
PubMed1983

The quantitative bioassay of nerve growth factor: use of frozen 'primed' PC12 pheochromocytoma cells.

A quantitative bioassay for nerve growth factor (NGF) has been previously described which employs PC13 rat pheochromocytoma cells. The basis of this assay is that rapid neurite regeneration by PC12 cells pretreated ('primed') with NGF for at least a week is quantitatively related to the amount of NGF present in the culture medium. The present experiments demonstrate that NGF-pretreated PC12 cells retain their priming for over a year when appropriately frozen and stored and that such frozen cells can be employed for the quantitative bioassay of NGF. The major advantage of this assay is that large numbers of cells can be primed at a time (in suspension culture) and stored in aliquots until future use. The newly described bioassay employs the frozen primed cells essentially as reagents and avoids the need to have freshly primed cultures on hand.

Adrenal Gland Neoplasms↗
PubMed1983

Ultrastructural effects of nerve growth factor on PC 12 pheochromocytoma cells in spinner culture.

PC12 pheochromocytoma cells treated with nerve growth factor (NGF) for two weeks in spinner cultures quickly begin to form processes after plating on an appropriate substrate, while cells freshly exposed to NGF in monolayer culture initiate neurite outgrowth only after a lag period of several days. The present ultrastructural studies indicate that PC 12 cells treated with NGF in spinner cultures do not form neurites, but do form short extensions comparable to those which have been reported within the first two days of exposure to NGF in monolayer cultures. These extensions contain organelles believed to be required for locomotion and for transport of cytoskeletal and membrane components and neurotransmitters. They also form bulbous distensions in which numerous chromaffin-type granules accumulate. These findings suggest that NGF may affect cells in spinner cultures by promoting development or activation of axonal transport mechanisms, and that the existence of these mechanisms may contribute to the neurite outgrowth which the cells exhibit when plated. NGF-treated PC 12 cells in spinner cultures do not accumulate the agranular synaptic-like vesicles, which are typically found in comparably treated monolayer cultures and which have been hypothesized to be sites of acetylcholine storage. These and other data demonstrate that attachment to a substrate can selectively modulate the responses of PC 12 cells to NGF.

Adrenal Gland Neoplasms↗
PubMed1983

Short-latency local actions of nerve growth factor at the growth cone.

Cultures of neurite-bearing pheochromocytoma (PC12) cells and of sympathetic neurons have been examined by time-lapse video microscopy. In the presence of nerve growth factor (NGF), the neurites of such cultures elongated and their growth cones changed geometry, via microspike and lamellipodial motion, on a time scale of minutes. Withdrawal of NGF caused process extension to cease and a progressive reduction in growth-cone area as a result of retraction of lamellipodia and microspikes. By approximately equal to 4 hr after NGF withdrawal, most neurite tips were smooth sided, devoid of conical expansions at their termini, and virtually immobile. Addition of NGF to cultures from which it had been withdrawn induced motion of microspikes and projections from the upper surface of growth cones within 2 min, while lamellipodial spreading and neurite reextension were induced after approximately equal to 20 min. For PC12 cells, these responses to replacement of NGF could not be mimicked by addition of dibutyryl cAMP (less than or equal to 2 mM) or the Ca2+ ionophore A23187 (less than or equal to 5 microM) to NGF-deprived cultures nor inhibited by the presence of EGTA (less than or equal to 2 mM) or calcium antagonists in the culture medium. Since neurite fragments formed by transection of processes of PC12 cells deprived of NGF responded to its replacement in a manner similar to intact neurites, it is concluded that the effects are focal to the neurite and growth cone and independent of the cell body. This influence of NGF on growth-cone shape and motility represents short-term local activation of this structure and has significance for control of neurite extension.

Adrenal Gland Neoplasms↗
PubMed1983

Regulation of a high molecular weight microtubule-associated protein in PC12 cells by nerve growth factor.

PC12 rat pheochromocytoma cells respond to nerve growth factor (NGF) protein by shifting from a chromaffin-cell-like phenotype to a neurite-bearing sympathetic-neuron-like phenotype. Comparison of the phosphoprotein patterns of the cells by SDS PAGE after various times of NGF treatment revealed a high molecular weight (Mr greater than or approximately 300,000) band whose relative intensity progressively increased beyond 2 d of NGF exposure. This effect was blocked by inhibitors of RNA synthesis and did not require neurite outgrowth or substrate attachment. The enhancement by NGF occurred in serum-free medium and was not produced by exposure to epidermal growth factor, insulin, dibutyryl cAMP, or dexamethasone. Several different types of experiments indicated that this phosphoprotein corresponds to a high molecular weight (HMW) microtubule-associated protein (MAP). These included cross-reactivity with antiserum against brain HMW MAPs, co-cycling with microtubules and co-assembly with tubulin in the presence of taxol. The affected species also co-migrated in SDS PAGE gels with brain MAP1 and, unlike MAP2, precipitated upon boiling. Studies with [35S]-methionine-labeled PC12 cells indicated that at least a significant proportion of this effect of NGF was due to increased levels of protein rather than to mere enhancement of phosphorylation. On the basis of the apparent effects of MAPs on the formation and stabilization of microtubules and of the importance of microtubules in production and maintenance of neurites, it is proposed that induction of a HMW MAP may be one of the steps in the mechanism whereby NGF promotes neurite outgrowth. Furthermore, these findings may lead to an understanding of the role of MAP1 in the nervous system.

Animals↗
PubMed1983

Nerve growth factor-inducible large external (NILE) glycoprotein: studies of a central and peripheral neuronal marker.

The PC12 clone of pheochromocytoma cells undergoes neuronal differentiation in the presence of nerve growth factor (NGF). Concomitant with this is a significant induction in the incorporation of radiolabeled fucose or glucosamine into a 230,000-dalton cell surface glycoprotein named the NGF-Inducible Large External, or NILE, glycoprotein (GP) (McGuire, J. C., L. A. Greene, and A. V. Furano (1978) Cell 15: 357-365). In the current studies NILE GP was purified from PC12 cells using wheat germ agglutinin-agarose affinity chromatography and SDS-polyacrylamide gel electrophoresis (PAGE). Polyclonal antisera were raised against purified NILE GP and were found to selectively immunoprecipitate a single 230,000-dalton protein from detergent extracts of PC12 cells metabolically labeled with either [3H]fucose, [3H]glucosamine, or [35S]methionine. These antisera stained the surfaces of PC12 cells by indirect immunofluorescence and were cytotoxic to PC12 cells in the presence of complement. Limited treatment of PC12 cells with either trypsin or pronase produced a fucosylated 90,000-dalton immunoreactive fragment of NILE GP which remained in the membrane. Using quantitative immunoelectrophoresis, the action of NGF on NILE GP was represent an increase in the amount of protein, rather than a selective increase in carbohydrate incorporation. Immunofluorescent staining of primary cell cultures and tissue whole mounts revealed that immunologically cross-reactive NILE GP appears to be expressed on the cell surfaces (somas and neurites) of most if not all peripheral and central neurons examined. Immunoprecipitation of radiolabeled cultures showed that the cross-reactive material had an apparent molecular weight by SDS-PAGE of 225,000 to 230,000 in the peripheral nervous system and 200,000 to 210,000 in the central nervous system. NILE-cross-reactive material was also found to a small extent on Schwann cell surfaces, but not at all on a variety of other cell types. These results suggest that immunoreactive NILE GP is distributed widely and selectively on neural cell surfaces.

Adrenal Gland Neoplasms↗
PubMed1983

Biochemical properties of the nerve growth factor-inducible large external (NILE) glycoprotein.

In the presence of nerve growth factor (NGF), PC12 pheochromocytoma cells undergo neuronal differentiation with a concomitant 3- to 5-fold increase in the specific level of an Mr = 230,000 cell surface component named the NGF-inducible large external, or NILE, glycoprotein. Antisera raised against NILE glycoprotein (NILE GP) purified from PC12 cells have been found to recognize most, if not all, neurons derived from the peripheral and central nervous systems. In the current studies several of the biochemical properties of NILE GP were investigated. NILE GP was found to be phosphorylated in NGF-treated and -untreated PC12 cells and in cultured rat sympathetic neurons. The phosphate moiety of NILE GP is almost completely alkali labile, suggesting that phosphoserine groups predominate. Immunoprecipitation experiments revealed that incorporation of [32P]phosphate into NILE GP relative to total PC12 cell phosphoprotein was not significantly altered at 12 and 24 hr of NGF treatment but was enhanced 3-fold after 7 days and up to 5-fold after 2 to 3 weeks of NGF exposure. These changes in phosphorylated NILE GP paralleled, and therefore appeared to be mainly a consequence of, the NGF-induced increase in total cellular levels of NILE GP. By two-dimensional gel analysis, anti-NILE GP selectively immunoprecipitated two NGF-inducible spots (apparent Mr = 230,000; pI = 6.4 to 6.6) from PC12 cells labeled with either [3H] fucose, [35S]methionine, or [32P]phosphate. Anti-NILE GP immunoprecipitated a single band (apparent Mr = 205,000) from extracts of rat brain labeled with [3H] glucosamine. This confirms the previously established apparent molecular weight difference between central and peripheral NILE GP cross-reactive material. When PC12 cells, cerebellar cultures, and cultured cerebral cortex were treated with tunicamycin and labeled with [35S]methionine, nonglycosylated bands each with Mr = 160,000 were immunoprecipitated, implying that the differences in the mobilities on sodium dodecyl sulfate gels of cross-reactive NILE GP from different tissues is due to variation in glycosylation rather than to large differences in apoprotein structure. Prolonged treatment of PC12 cells with trypsin produced an immunoreactive fragment of NILE GP of apparent Mr = 28,000 that was phosphorylated but not glycosylated, and that remained in the membrane. NILE GP remained predominantly membrane associated under a variety of aqueous extraction conditions, suggesting that it is an integral membrane protein.

Animals↗