cAMP-induced Differentiation Research Articles

Increased expression and secretion of r-Gsp protein, rat counterpart of complement C1s precursor, during cyclic AMP-induced differentiation in rat C6 glioma cells

The gene, termed r-gsp, was originally isolated during identification of differentiation-associated molecules in rat C6 glial cells. Its mRNA expression was markedly increased during cAMP-induced glial cell differentiation. The deduced amino acid sequence of r-gsp was homologous to those of complement C1s precursors of hamsters and humans. In the present study, we raised anti-peptide antibody against r-Gsp protein and analyzed its change during cAMP-induced differentiation. The 90-kDa r-Gsp protein increased time-dependently and reached the maximal level (∼7.6-fold increase) at 24 h in response to dibutyryl cyclic AMP (dbcAMP) and theophylline. Moreover, it was secreted into the medium and then was cleaved to form disulfide-linked fragments, one of which was 30 kDa, similar to C1s, suggesting its processing in the extracellular space. In fact, the partially purified r-Gsp from culture medium was cleaved by active human C1r to form a 30-kDa polypeptide. Moreover, secreted r-Gsp protein cleaved human C4α to yield C4α′ and associated with human serum C1-esterase inhibitor, strongly suggesting that r-Gsp protein is rat C1s. However, in C6 cells overexpressing r-Gsp, their morphology and proliferation rate were similar to those in parent C6 cells. These results suggest that r-Gsp protein could not induce glial differentiation alone, and suggest that r-Gsp protein was secreted as a proenzyme and processed in culture medium. Its possible role in glial cell differentiation will be discussed.

Cyclic AMP induces activation of extracellular signal-regulated kinases in HL-60 cells: role in cAMP-induced differentiation

It is well known that elevated intracellular cAMP induces growth arrest and the differentiation of HL-60 cells to neutrophil-like cells. The present study was designed to assess the regulation of the extracellular signal-regulated kinase (ERK) pathway by cAMP and its association with differentiation in HL-60 cells. We found that 8-bromoadenosine-3′,5′- cyclic-monophosphate (8Br-cAMP)-induced the activation of ERK and mitogen-activated protein kinase (MEK), but inhibited B-Raf kinase via a protein kinase A (PKA)-mediated mechanism. Prolonged exposure to 8Br-cAMP increased the phorbol 12-myristate 13-acetate (TPA)-stimulated superoxide generation and CD14 expression that characterize the differentiation phenotype, which was blocked by MEK-1 inhibitor. These data suggest that cAMP-induced ERK activation is essential for the differentiation of HL-60 cells, independently of B-Raf.

Altered expression of genes regulating cell growth, proliferation, and apoptosis during adenosine 3',5'-cyclic monophosphate-induced differentiation of neuroblastoma cells in culture.

An elevation of the intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) induces terminal differentiation in neuroblastoma (NB) cells in culture; however, genetic alterations during differentiation have not been fully identified. To investigate this, we used Mouse Genome U74A microarray containing approximately 6000 functionally characterized genes to measure changes in gene expression in murine NB cells 30 min and 4, 24, and 72 h after treatment with cAMP-stimulating agents. Based on the time of increase in differentiated functions and their status (reversible versus irreversible) after treatment with cAMP-stimulating agents, the induction of differentiation in NB cells was divided into three distinct phases: initiation (about 4 h after treatment when no increase in differentiated functions is detectable), promotion (about 24 h after treatment when an increase in differentiated functions occurs, but they are reversible upon the removal of cAMP), and maintenance (about 72 h after treatment when differentiated functions are maximally expressed, but they are irreversible upon the removal of cAMP). Results showed that alterations in expression of genes regulating cell growth, proliferation, apoptosis, and necrosis occurred during cAMP-induced differentiation of NB cells. Genes that were upregulated during the initiation, promotion, or maintenance phase were called initiators, promoters, or maintainers of differentiation. Genes that were downregulated during the initiation, promotion, or maintenance phase were called suppressors of initiation, promotion, or maintenance phase. Genes regulating growth may act as initiators, promoters, maintainers, or suppressors of these phases. Genes regulating cell proliferation may primarily act as suppressors of promotion. Genes regulating cell cycle may behave as suppressors of initiation or promotion, whereas those regulating apoptosis and necrosis may act as initiators or suppressors of initiation or promotion. The fact that genetic signals for differentiation occurred 30 min after treatment with cAMP, whereas cell-cycle genes were downregulated at a later time, suggests that decision for NB cells to differentiate is made earlier and not at the cell-cycle stage, as commonly believed.

Inhibitory effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on cAMP-induced differentiation of rat C6 glial cell line.

Dioxin is suspected to cause adverse effects on the development of the central nervous system (CNS). To investigate the neurotoxic effects of dioxin on the differentiation of astrocytes, rat C6 glial cell line was used as a model, because these cells are induced to express astrocyte markers and to change the cell morphology toward an astrocytic phenotype by increasing intracellular cAMP levels. When C6 cells were simultaneously exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and N(6),O(2')-dibutylyl cAMP (dbcAMP), the expression of cytochrome P-450 1A1 (CYP1A1) was dramatically increased, and the expression of aryl hydrocarbon receptor (AhR) was moderately decreased in a dose-dependent manner. In addition, extension of astrocytic processes was inhibited by 1 nM TCDD that did not reduce cell viability. TCDD also inhibited the induction of glial fibrillary acidic protein (GFAP) expression in a dose-dependent manner, until the end of a 72-hr exposure period. This inhibition was restored by the addition of an antagonist of AhR, alpha-naphthoflavone. These results indicate that TCDD inhibits astrocytic differentiation of C6 cells, which may be mediated by an AhR-dependent pathway.

Proteasome activity is critical for the cAMP-induced differentiation of neuroblastoma cells.

1. The ubiquitin-proteasome pathway is involved in a variety of cellular functions in mammalian cells. The role of proteasome, however, in the course of cell differentiation is not well characterized. We hypothesized that proteasome activity might be essential during neuronal cell differentiation. 2. To investigate the role of proteasome during neuronal differentiation, we made use of a murine neuroblastoma cell line (NBP2) that terminally differentiates into mature neurons upon elevation of the intracellular level of adenosine 3',5'-cyclic monophosphate (cAMP). To monitor proteasome activity in NBP2 cells, we integrated an expression cassette for a short-lived green fluorescent protein (d2EGFP) into these cells, which were designated as NBP2-PN25. When NBP2-PN25 cells were treated with a proteasome inhibitor, lactacystin or MG132, a dose-dependent increase in the constitutive levels of d2EGFP expression was detected. 3. We also found that proteasome inhibition by lactacystin during the cAMP-induced differentiation of NBP2-PN25 cells triggered cell death. Both lactacystin and cAMP induction reduced the expression of mRNA for the differentiation-associated genes, such as N-myc and cyclin B1. While cAMP-inducing agents decreased the level of N-myc and cyclin B1 proteins, lactacystin increased the level of these proteins. 4. Our data suggest that a reduced level of N-myc and cyclin B1 proteins is critical to commence differentiation, and this can be blocked by a proteasome inhibitor, leading to cell death. Concomitant induction of differentiation and proteasome inhibition, may, therefore, be potentially useful for the treatment of human neuroblastomas.

Nucleoside Diphosphate Kinase β (Nm23-R1/NDPKβ) Is Associated with Intermediate Filaments and Becomes Upregulated upon cAMP-Induced Differentiation of Rat C6 Glioma

Nucleoside diphosphate kinases (Nm23/NDPK) are enzymes functional in cell proliferation, differentiation, development, tumor progression, and metastasis. Nevertheless, no consensus exists about the molecular mechanism by which Nm23/NDPK isoforms exert their role in these processes. We investigated the expression of the rat Nm23-R1/NDPKβ and Nm23-R2/NDPKα isoforms, homologues of the human Nm23-H1/NDPK A and Nm23-H2/NDPK B proteins, respectively, upon cAMP-induced differentiation of rat C6 glioma cells and demonstrated a differential interaction with intermediate filaments. Semiquantitative RT-PCR, immunoblotting, and flow cytometry showed a constitutive expression of both Nm23 isoforms. After induction of differentiation in C6 cells with cAMP analogs or isoproterenol, a dose-dependent 2- and 2.5-fold upregulation of the Nm23-R1 mRNA and protein, respectively, was observed. In contrast, the expression of Nm23-R2 remained unchanged. Localization of both isoforms with confocal laser scanning microscopy demonstrated a punctate reticular staining pattern for both Nm23 isoforms in the cytosol and processes of the cells which was particularly intense in the perinuclear region. In addition, while Nm23-R2 was colocalized and coimmunoprecipitated with vimentin in nondifferentiated cells, both isoforms were associated with GFAP in differentiated cells. The significance of these findings in relation to a possible function of Nm23 isoforms in cell proliferation, differentiation, and tumor-associated mechanisms is discussed.

Staurosporine enhances cAMP-induced expression of neural-specific gene VGF and tyrosine hydroxylase

Cyclic AMP transiently stimulates transcription of specific genes in the nervous system, including neural-specific gene VGF. The simultaneous presence of staurosporine (SSP) caused a progressive, rather than transient, increase in VGF mRNA levels during the cAMP-induced differentiation of human neuroblastoma cells. This steady increase does not appear to be due to increased stability of VGF mRNA. This synergy between cAMP and SSP was also observed at the protein level for tyrosine hydroxylase. These findings suggest the presence of cellular machinery that counteracts the decline of cAMP-induced gene expression.

Microphthalmia gene product as a signal transducer in cAMP-induced differentiation of melanocytes.

Melanocyte differentiation characterized by an increased melanogenesis, is stimulated by alpha-melanocyte-stimulating hormone through activation of the cAMP pathway. During this process, the expression of tyrosinase, the enzyme that controls melanin synthesis is upregulated. We previously showed that cAMP regulates transcription of the tyrosinase gene through a CATGTG motif that binds microphthalmia a transcription factor involved in melanocyte survival. Further, microphthalmia stimulates the transcriptional activity of the tyrosinase promoter and cAMP increases the binding of microphthalmia to the CATGTG motif. These observations led us to hypothesize that microphthalmia mediates the effect of cAMP on the expression of tyrosinase. The present study was designed to elucidate the mechanism by which cAMP regulates microphthalmia function and to prove our former hypothesis, suggesting that microphthalmia is a key component in cAMP-induced melanogenesis. First, we showed that cAMP upregulates the transcription of microphthalmia gene through a classical cAMP response element that is functional only in melanocytes. Then, using a dominant-negative mutant of microphthalmia, we demonstrated that microphthalmia is required for the cAMP effect on tyrosinase promoter. These findings disclose the mechanism by which cAMP stimulates tyrosinase expression and melanogenesis and emphasize the critical role of microphthalmia as signal transducer in cAMP-induced melanogenesis and pigment cell differentiation.

A Putative G-Protein-Coupled Receptor, H218, Is Down-regulated during the Retinoic Acid-Induced Differentiation of F9 Embryonal Carcinoma Cells

We have previously cloned a novel guanine nucleotide-binding protein (G-protein)-coupled receptor, H218, that has sequence similarity to a lysophosphatidic acid receptor, edg2. We present here Northern analysis indicating that the H218 mRNA is expressed in undifferentiated F9 embryonal carcinoma cells. The H218 message is down-regulated and its stability is decreased during retinoic acid- and dibutyryl cAMP-induced differentiation. Treatment by various receptor-selective retinoids indicated that retinoic acid receptor β or γ signaling, but not retinoid X receptor activation, is required for the down-regulation of H218 mRNA. Activation of the H218 receptor may contribute to the phenotype of undifferentiated F9 embryonal carcinoma cells.

Dominant negative ATF1 blocks cyclic AMP-induced neurite outgrowth in PC12D cells.

Extension of the neuronal process is a crucial step for establishment of the neuronal network. As CREB preferentially forms heterodimers with ATF1 in PC12D cells, we examined the roles of the CREB/ATF1 heterodimer on cyclic AMP (cAMP)-induced neurite extension, using originally constructed ATF1RL, which has a point mutation at the DNA binding domain of ATF1. Transient expression of ATF1RL suppressed the protein kinase A/CREB-induced expression of the CRE reporter gene as expected. Treatment with forskolin elicited a relatively poor mRNA induction for immediate early genes in PC12D-ATF1 RL cells, a PC12D cell line stably expressing ATF1RL, in comparison with the parental PC12D cells. Furthermore, the PC12D-ATF1RL cells were proved to be defective at cAMP-induced neurite outgrowth. In contrast, both the gene expression and the differentiation after nerve growth factor treatment noted in PC12D-ATF1RL cells were at the same levels as those in the parental cells. These data provide us the first evidence that links CREB/ATF1 to the cAMP-induced differentiation of PC12 cells.

Differential mRNA Expression of Phospholipase D (PLD) Isozymes during cAMP-Induced Differentiation in C6 Glioma Cells

GTPγS-dependent phospholipase D (PLD) activity time-dependently increased during differentiation of rat C6 glioma cells to astrocytic phenotypes induced by dibutyryl cyclic AMP (dbcAMP)/theophylline. The changes in PLD mRNA level were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) method using the degenerate primers designed based on two conserved amino acid sequences in PLDs of human and yeast. The amplified three DNA fragments (tentatively termed as rPLDa, b, and c) contained the conserved regions present in PLDs of various organisms. RT-PCR using non-degenerate primers showed that rPLDa mRNA increased within 12h following treatment with dbcAMP, reaching a broad plateau and then returned to the initial level at 48h. In contrast, the level of rPLDb mRNA showed a concurrent decrease. rPLDc decreased in a time-dependent manner. These results suggest that the expression of PLD mRNAs are differentially regulated during differentiation in C6 glioma cells.

Increased expression of ubiquitin during adenosine 3',5'-cyclic monophosphate-induced differentiation of neuroblastoma cells in culture.

The role of ubiquitin in proliferation and differentiation of nerve cells has not been studied. An elevation of the intracellular level of adenosine 3',5'-cyclic monophosphate (cAMP) in neuroblastoma cells induces terminal differentiation in these cells. Therefore, in this study we investigated the changes in the level and subcellular distribution of ubiquitin during proliferation and differentiation of neuroblastoma cells. Prostaglandin E1, a stimulator of adenylate cyclase, plus beta-carotene, and 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724), an inhibitor of cyclic nucleotide phosphodiesterase, plus beta-carotene were used to induce terminal differentiation in > 90% of neuroblastoma cells. Changes in ubiquitin level were studied by immunofluorescent staining using either a mouse monoclonal antibody or a rabbit polyvalent antibody to ubiquitin. Results showed that the dividing neuroblastoma cells contained very low levels of ubiquitin localized primarily in the cytoplasm. The intensity of cytoplasmic staining for ubiquitin markedly increased during cAMP-induced differentiation of neuroblastoma cells, being the highest at 4 days after treatment. The neurites of these differentiated cells were also stained, but the nuclei were not. We propose a hypothesis that higher levels of cytoplasmic ubiquitin are needed during cAMP-induced differentiation of neuroblastoma cells for the removal of proteins responsible for cell proliferation through rapid degradation and/or inhibition of transcription, later leading to terminal differentiation.

IgG-induced Ca 2+ oscillations in differentiated U937 cells; a study using laser scanning confocal microscopy and co-loaded Fluo-3 and Fura-Red fluorescent probes

We have investigated, at the single cell level, intracellular Ca 2+ ([Ca 2+] i) modulations triggered by the high affini71 receptor for IgG, FcγRI, in the monocytic cell line, 0937. Cells were co-loaded with the Ca 2+-sensitive dyes, Fluo-3 and Fura-Red, by incubation with their acetoxymethyl (AM) esters and confocal ratio imaging was used to monitor the [Ca 2+] i changes induced by antibody cross-linking of IgG-loaded FcγRI. A single Ca 2+ spike was observed in 81% of untreated cells whereas dibutyryl CAMP-induced differentiation into a more macrophage cell type resulted in a sub-population of cells (44%) responding to receptor cross-linking with calcium oscillations. This change in calcium signalling may explain the difference in functional responses triggered by FcγRI in monocytes and macrophages. Analysis of the Fluo-3 and Fura-Red fluorescence, after AM-ester loading, showed that both dyes have similar photobleach rates and intracellular localization allowing compensation for shifts in focal plane, dye photobleaching and non-uniformity of dye loading. In addition, because the binding kinetics of both dyes are equivalent, accurate temporal information can be gained about [Ca 2+] changes. There are, however, two major problems with this dual indicator technique. Firstly, loading from AM esters results in considerable variation between cells in the intracellular concentration ratio of the two dyes, making calibration difficult. Secondly, the fluorescence ratio, Fluo-3/Fura-Red, behaves non-linearly at Ca 2+ concentrations less than ∼500 nM and comparison with Fura-2-loaded single cell photometry studies suggests there is considerable amplitude distortion of the signal when the ratios are displayed on a linear scale. These problems may considerably limit the application of Fluo-3/Fura-Red ratiometric measurements.

Involvement of p53 Expression in cAMP-Mediated Apoptosis in Immortalized Granulosa Cells

In the accompanying paper we described the induction of apoptosis by extended cyclic AMP (cAMP)-mediated signals in primary granulosa cells and the reduction in this process in transformed cells expressing SV40 T antigen. In the present work, we examined the effect of overexpression of either wild-type or mutant p53 on cAMP-mediated apoptosis in steroidogenic granulosa cell lines transfected with SV40 DNA together with the Ha- ras oncogene and a temperature-sensitive variant of p53, p53val135. In cell lines expressing low amounts of T antigen and high amounts of p53val135, growth arrest was induced by transferring the cells from 37.5° to 32°C, a temperature which allows the manifestation of the wild-type phenotype of p53 and the induction of the WAF1 gene. While nonstimulated cells showed only a very modest apoptotic process, rapid and massive apoptosis was evident in cells stimulated by forskolin at 32°C. The presence of serum could delay, but not abolish, this phenomenon. Progesterone production in such cells treated with cAMP was significantly higher at 32°C than at 37.5°C, suggesting that wild-type p53 can also enhance granulosa cell differentiation. Furthermore, at least at early stages, apoptosis is correlated with increased cell differentiation. On the other hand, in lines expressing high amounts of T antigen and low amounts of p53, neither an increase in cAMP-induced differentiation nor massive apoptosis was seen at 32°C. These findings demonstrate that wild-type p53 can cooperate with cAMP-generated signals in the induction of steroidogenesis and of programmed cell death in granulosa cells.

Role of phospholipase-D and phosphatidic acid in mediating gonadotropin-releasing hormone-induced inhibition of preantral granulosa cell differentiation.

The activation of phospholipase-D (PLD) was previously implicated in mediating the differentiative action of GnRH on preovulatory granulosa cells. The activation of PLD and the action of its product, phosphatidic acid (PA), were further studied in preantral granulosa cells, where GnRH exerts an antidifferentiative effect. A GnRH receptor agonist (GnRH-A) activated PLD in the cells, causing a sustained elevation of phosphatidylethanol and a transient increase in cellular PA levels. PLD was also activated by 12-O-tetradecanoylphorbol-13-acetate (TPA). Both GnRH-A and TPA inhibited FSH-induced production of progesterone, a marker of granulosa cell differentiation. D,L-Propranolol, which elevates cellular PA by inhibiting its degradation, mimicked the antidifferentiative action of GnRH-A and TPA in a dose-dependent manner. Addition of PA similarly inhibited FSH-induced progesterone production in a dose-dependent manner. The effect of forskolin, which mimics the steroidogenic effect of FSH by elevating intracellular levels of cAMP, could also be suppressed by GnRH-A and PA. FSH- and cAMP-induced differentiation of preantral granulosa cells is characterized by cell rounding and breakdown of actin filament bundles. This effect is inhibited by GnRH-A and TPA as well as PA. It is concluded that activation of PLD and the resultant production of PA could mediate the antidifferentiative action of GnRH in preantral granulosa cells. Moreover, GnRH-induced PLD-generated signals counteract the FSH-induced cAMP-dependent signals that modulate the organization of the actin cytoskeleton characteristic of steroidogenic cells.

Changes in intracellular localization of proteasomes in immortalized ovarian granulosa cells during mitosis associated with a role in cell cycle control.

We describe the isolation and characterization of proteasomes from recently established immortalized ovarian granulosa cell lines and their intracellular distribution during mitosis and during cAMP-induced differentiation, as revealed by immunofluorescence microscopy. In interphase, proteasomes were localized in small clusters throughout the cytoplasm and the nuclear matrix. In prophase, a substantial increase in proteasomal staining was observed in the perichromosomal area. A dramatic increase occurred in metaphase and in early anaphase; the chromosomes remained unstained. In late anaphase, intensive staining remained associated mainly with the spindle fibers. In telophase and early interphase of the daughter cells, intensive staining of proteasomes persisted in the nuclei. In contrast, in cells stimulated to differentiate by forskolin, which substantially elevates intracellular cAMP in these cell lines, only a weak staining of proteasomes was revealed in both the nucleus and the cytoplasm. Double staining of nondividing cells with antibodies to proteasomes and to tubulin did not show colocalization of proteasomes and microtubules. In contrast, dividing cells show a preferential concentration of proteasomes around spindle microtubules during metaphase and anaphase. The observed spatial and temporal distribution pattern of proteasomes during mitosis is highly reminiscent of the behavior of cyclins [Pines, J. & Hunter, T. (1991) J. Cell Biol. 115, 1-17]. Since proteasome accumulation appears to coincide with disappearance of cyclins A and B1 from the spindle apparatus, it is suggested that proteasomes may play a role in termination of mitosis by degrading the cyclins, which act as regulatory elements.

Selective upregulation of the NCAM-B isoform in cultured cerebral astrocytes following cAMP-induced differentiation

Selective upregulation of the NCAM-B isoform in cultured cerebral astrocytes following cAMP-induced differentiation

P-252 - Alteration of Na, K-pump activity in dibutylyl cAMP-induced differentiation of PCl2h cells

P-252 - Alteration of Na, K-pump activity in dibutylyl cAMP-induced differentiation of PCl2h cells

Effects of growth medium and cyclic AMP analogues on the cAMP-induced differentiation of F9 teratocarcinoma cells

F9 teratocarcinoma cells differentiate into parietal endodermlike cells when treated with retinoic acid (RA) and cyclic AMP (cAMP). We have previously found that F9 cells can be induced to differentiate by treatment with cAMP in the absence of RA. In the course of determining why other investigators had failed to observe cAMP-induced differentiation, we found that the growth medium played an important role in determining the response of F9 cells to differentiating agents. When F9 cells were grown in minimal essential medium (MEM) and treated with either 8-bromo-cAMP (8BrcA) + 1-methyl, 3-isobutylxanthine (MIX), or dibutyryl cAMP (DBcA) + theophylline (T), they differentiated to parietal endodermlike cells without any requirement for exogenous RA. However, when F9 cells were grown in Dulbecco's modified Eagle's medium (DME), DBcA/T failed to induce differentiation alone and required exogenous RA to induce formation of parietal endoderm-like cells. 8BrcA/MIX alone was still able to induce some differentiation, although the extent was not as great as those cells grown in MEM. These results could not be explained by the different growth-promoting properties of the two culture media because there was no difference in the doubling time of F9 cells grown in either medium. Likewise, RA and cAMP both inhibited growth to the same extent in either medium. Inasmuch as almost all published reports on F9 cell differentiation have used DME as a growth medium, and RA with or without DBcA/T as the differentiating agents, these studies would not have had the appropriate conditions to detect the cAMP-induced differentiation of F9 cells.

Identification of Two Distinct Isoforms of the Guanine Nucleotide Binding Protein Go in Neuroblastoma × Glioma Hybrid Cells: Independent Regulation During Cyclic AMP‐Induced Differentiation

Three distinct antipeptide antisera generated against synthetic peptides that represent parts of the primary sequence of the alpha-subunit of the (pertussis toxin-sensitive) guanine nucleotide binding protein G0 were used in two-dimensional immunoblots of membranes of neuroblastoma X glioma (NG108-15) cells. Each antiserum identified two distinct polypeptides of some 39 kDa. These had apparent isoelectric points of 5.5 and 5.8. Differentiation of NG108-15 cells in response separately to dibutyryl cyclic AMP (cAMP), 8-bromo cAMP, forskolin, and prostaglandin E1 produced elevated levels of G0 alpha, as has previously been noted in one-dimensional immunoblots. Two-dimensional analysis demonstrated that the cAMP-induced increases in levels of G0 alpha were only of the more acidic isoform. The two isoforms were both substrates for pertussis toxin-catalysed ADP-ribosylation and did not appear to represent differentially phosphorylated forms of the same polypeptide. Separation of the two forms of G0 alpha could be achieved in one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis when 4 M deionized urea was included in the resolving gel. The more slowly migrating band was the acidic form and corresponded exactly in mobility with the major form of G0 from both rat and mouse brain. There was no equivalent in brain of the more rapidly migrating form of G0 from the cells. In agreement with the data from two-dimensional gels, only the more slowly migrating form was expressed in considerably higher amounts following cAMP-induced differentiation of NG108-15 cells. Of these two forms of "G0," the acidic species is equivalent to G0 from brain, but the basic form is not identical with G0*, which has been purified from bovine brain.