Aberrant nucleocytoplasmic localization of the retinoblastoma tumor suppressor protein in human cancer correlates with moderate/poor tumor differentiation.

Inactivation of the retinoblastoma (RB) tumor suppressor pathway, via elevated cyclin-dependent kinase (CDK) activity, is observed in majority of human cancers. Since CDK deregulation is evident in most cancer cells, pharmacological CDK inhibition has become an attractive therapeutic strategy in oncology. We recently showed that an oncogenic CDK4(R24C) mutation alters the subcellular localization of the normally nuclear RB phosphoprotein. Here, using 71 human cancer cell lines and over 300 primary human cancer tissues, we investigated whether changes in RB subcellular localization occur during human cancer progression. We uncover that diverse human cancers and their derived cell lines, particularly those with poor tumor differentiation, display significant cytoplasmic mislocalization of ordinarily nuclear RB. The nucleocytoplasmically distributed RB was derived via CDK-dependent and Exportin1-mediated nuclear export. Indeed, cytoplasmically mislocalized RB could be efficiently confined to the nucleus by pharmacologically reducing CDK activity or by inhibiting the Exportin1-mediated nuclear export pathway. Our observations uncover a post-translational CDK-dependent mechanism of RB inactivation and suggest that cytoplasmically localized RB may harbor a tumor promoting function. We propose that RB inactivation, via aberrant nucleocytoplasmic transport, may disrupt normal cell differentiation programs and accelerate the cancer process. These results are evidence that tumor cells modulate the protein transport machinery thereby making the protein transport process a viable therapeutic target.

Smad3 deficiency inhibits v-ras-induced transformation by suppression of JNK MAPK signaling and increased farnesyl transferase inhibition.

The ability of transforming growth factor-beta (TGF-beta) to modulate various effects on distinct cell lineages has been a central feature of its multi-faceted nature. The purpose of this study was to access the effects of deletion of a key TGF-beta signal transducer, Smad3, on MAPK activation and v-Ras(Ha)-transformation of primary mouse embryonic fibroblasts (MEFs). We observe reduced TGF-beta1 and v-ras(Ha) mediated activation of the JNK and ERK MAPK pathway upon ablation of Smad3. Further, Smad3-deficient MEFs demonstrate resistance to v-ras(Ha)-induced transformation while the absence of Smad3 results in increased inhibition of farnesyl transferase activity. Taken together, these observations demonstrate that the absence of Smad3 protects fibroblasts from oncogenic transformation by (i) augmenting farnesyl transferase inhibition and (ii) suppressing the Ras-JNK MAPK pathway. These results provide new insights into the molecular mechanisms involved in v-Ras(Ha) oncogene-induced mesenchymal phenotypic transformation.

Hematopoietic cytokine receptor signaling.

Hematopoiesis is the cumulative result of intricately regulated signaling pathways that are mediated by cytokines and their receptors. Proper culmination of these diverse pathways forms the basis for an orderly generation of different cell types. Recent studies conducted over the past 10-15 years have revealed that hematopoietic cytokine receptor signaling is largely mediated by a family of tyrosine kinases termed Janus kinases (JAKs) and their downstream transcription factors termed STATs (signal transducers and activators of transcription). Aberration in these pathways, such as that caused by the recently identified JAK2V617F mutation, is an underlying cause for diseases such as leukemias and other myeloproliferative disorders. This recent discovery, when coupled with the fact that STATs are activated by oncoproteins such as BCR-ABL, underscores the importance of the JAK-STAT pathway in both normal cellular development and disease states.

Granulocyte colony-stimulating factor-induced upregulation of Jak3 transcription during granulocytic differentiation is mediated by the cooperative action of Sp1 and Stat3.

We previously demonstrated that Jak3 is a primary response gene for G-CSF and ectopic overexpression of Jak3 can accelerate granulocytic differentiation of normal mouse bone marrow cells induced by G-CSF and GM-CSF. To gain insight into the regulation of G-CSF-induced transcription of Jak3, we constructed deletion and linker scanning mutants of the Jak3 promoter sequences and performed luciferase reporter assays in the murine myeloid cell line 32Dcl3, with and without G-CSF stimulation. These experiments showed that mutation of a -67 to -85 element, which contained a putative Sp1 binding site, or mutation of a -44 to -53 GAS element resulted in a marked reduction of Jak3 promoter activity. Electrophoretic mobility shift assays revealed that Sp1 and Stat3 present in nuclear lysates of 32Dcl3 cells stimulated with G-CSF can bind to the -67 to -85 element and -44 to -53 GAS element, respectively. In addition, cotransfection of a constitutively active mutant of Stat3 along with a Jak3 promoter/luciferase reporter resulted in enhanced Jak3 promoter activity. Together, these results demonstrate that activation of Jak3 transcription during G-CSF- induced granulocytic differentiation is mediated by the combined action of Sp1 and Stat3, a mechanism also shown to be important in IL-6-induced monocytic differentiation.

Janus kinases: components of multiple signaling pathways.

Cytoplasmic Janus protein tyrosine kinases (JAKs) are crucial components of diverse signal transduction pathways that govern cellular survival, proliferation, differentiation and apoptosis. Evidence to date, indicates that JAK kinase function may integrate components of diverse signaling cascades. While it is likely that activation of STAT proteins may be an important function attributed to the JAK kinases, it is certainly not the only function performed by this key family of cytoplasmic tyrosine kinases. Emerging evidence indicates that phosphorylation of cytokine and growth factor receptors may be the primary functional attribute of JAK kinases. The JAK-triggered receptor phosphorylation can potentially be a rate-limiting event for a successful culmination of downstream signaling events. In support of this hypothesis, it has been found that JAK kinase function is required for optimal activation of the Src-kinase cascade, the Ras-MAP kinase pathway, the PI3K-AKT pathway and STAT signaling following the interaction of cytokine/interferon receptors with their ligands. Aberrations in JAK kinase activity, that may lead to derailment of one or more of the above mentioned pathways could disrupt normal cellular responses and result in disease states. Thus, over-activation of JAK kinases has been implicated in tumorigenesis. In contrast, loss of JAK kinase function has been found to result in disease states such as severe-combined immunodeficiency. In summary, optimal JAK kinase activity is a critical determinant of normal transmission of cytokine and growth factor signals.

Low extracellular Ca(2+) activates a transient Cl(-) current in chicken ovarian granulosa cells.

The effects of low Ca(2+) on ion currents in hen ovarian granulosa cells were examined. A fast activating and inactivating transient outward current (TOC) and a slowly activating outward current (SOC) could be observed. In the presence of normal Ca(2+) concentration (2. 5 mM) and with a holding potential of -80 mV, SOC was activated in all cells with command pulses more positive than -20 mV. In 2.5 mM Ca(2+), TOC appeared in 10% of cells at the command pulse of +80 mV and in 60-85% of cells at +100 to +120 mV. In low-Ca(2+) solution and command potential of +80 mV (holding potential of -80 mV), the amplitude of TOC was enhanced in cells that expressed it in normal Ca(2+), and TOC appeared in 43% of the cells that did not express it initially in normal Ca(2+). At both normal and low Ca(2+) levels, TOC decreased as the holding potential became more positive. TOC was reduced in Cl(-)-deficient solution and in the presence of 5-nitro-2-(3-phenylpropylamino)benzoic acid, a Cl(-) channel blocker. These findings suggest that chicken granulosa cells express a Ca(2+)-inactivated TOC carried by Cl(-). This current may serve as a signal for some of the reduced metabolic functions of granulosa cells associated with Ca(2+) deficiency.

Ras/MEK/ERK Up-regulation of the fibroblast KCa channel FIK is a common mechanism for basic fibroblast growth factor and transforming growth factor-beta suppression of myogenesis.

The 10T1/2-MRF4 fibroblast/myogenic cell system was used to address the following interrelated questions: whether distinct signaling pathways underlie myogenic inhibition by basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-beta; which of these pathways also up-regulates the fibroblast intermediate conductance calcium-activated potassium channel, FIK, a positive regulator of cell proliferation; and whether FIK up-regulation underlies some or all myogenic inhibitory signaling events. The results show that myogenic inhibition in 10T1/2-MRF4 cells, by both bFGF and TGF-beta, requires activation of the Ras/mitogen-activated protein (MAP) kinase/MAP kinase-ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, and resultant FIK up-regulation. We show that FIK is instrumental in MEK-dependent suppression of acetylcholine receptor channel expression but that MEK activation and FIK up-regulation are not essential to suppression of myosin heavy chain and myotube formation. These data indicate that Ras/MEK/ERK induction of FIK is pivotal to regulation of certain myogenic events by both receptor tyrosine kinases and TGF-beta receptor, and this is also the first demonstration of chronic FIK up-regulation by the TGF-beta receptor family. Furthermore, the results define the physiologic signaling requirements for growth factor-stimulated FIK up-regulation, whereas previous work has concentrated on constitutive FIK up-regulation in cells stably transfected with oncoprotein signaling molecules. This study, together with earlier work showing that FIK positively regulates cell proliferation, establishes this member of the IK channel family as a multifunctional, growth factor-regulated signaling molecule.

The growth regulatory fibroblast IK channel is the prominent electrophysiological feature of rat prostatic cancer cells.

Physiological effectors for mitogenic cell growth control remain to be determined for mammalian tumor cells, particularly those derived from prostatic tissue. One such effector for mitogenic Ras/MAPK signaling in fibroblasts is an intermediate-conductance, calcium-activated potassium channel (FIK). In this study patch-clamp electrophysiology was used to show that both AT2.1 and MatLyLu rat prostate cancer cell lines express high levels of a current identified as FIK, based on the following criteria: activation by elevation of intracellular calcium, voltage independence, potassium selectivity, and block by charybdotoxin (ChTX) and the Stichodactyla helianthus potassium channel neurotoxin (StK). FIK current densities in AT2.1 and MatLyLu cells were comparable to the high levels seen in fibroblasts transfected with oncogenic Ras or Raf, suggesting hyperactivity of the Ras/MAPK pathway in prostatic cancer cells. Voltage-gated sodium current was present in most MatLyLu cells but absent from AT2.1 cells, and all AT2.1 cells had voltage-gated potassium currents. Thus, FIK is the main electrophysiological feature of rat prostatic cancer cells as it is for mitogenically active fibroblasts, suggesting it may play a similar growth regulatory role in both.

Cell cycle control of pancreatic beta cell proliferation.

Diabetes mellitus ensues as a consequence of the body's inability to respond normally to high blood glucose levels. The onset of diabetes is due to several pathological changes, which are a reflection of either the inability of the pancreatic beta cells to secrete sufficient insulin to combat the hyperglycemia or a state of insulin resistance in target tissues. However, the significance of changes in beta cell mass and decreased beta cell proliferation or growth in progression of diabetes has been under-appreciated. Beta cells, like all other cells of our body are under the regulatory checks and balances enforced by changes in cell cycle progression. However, very little is known regarding the key components of the cell cycle machinery regulating cell cycle control of beta cells. Knowledge of key elements involved in cell cycle regulation of beta cells will go a long way in improving our understanding of the replication capacity and developmental biology of beta cells. This information is essential for us to design new approaches that can be used to correct beta cell deficiency in diabetes. This review focuses on the current knowledge of factors important for proliferation of beta cells and proposes a cell cycle model for regeneration of the beta cell population lost or reduced in diabetes.

The fibroblast intermediate conductance K(Ca) channel, FIK, as a prototype for the cell growth regulatory function of the IK channel family.

The fibroblast intermediate conductance, calcium-activated potassium channel (FIK) is proposed here as a functional prototype for other IK channels which to date have undefined physiologic actions. FIK pharmacology in the 10T1/2-MRF4 myogenic fibroblast cell line was determined: to define the relationship of FIK to other IKs; to confirm a physiologic role for FIK; and, thus develop a hypothesis about IK channel family function. Whole cell patch-clamp electrophysiology was used to determine K(0.5) values for FIK block by the structurally related peptides charybdotoxin (ChTX) (7 nm) and iberiotoxin (IbTX) (536 nm), and a new unrelated FIK inhibitor, Stichodactyla toxin (StK) (85 nm). Peptide pharmacology for FIK was consistent with that of recently cloned IKs. ChTX and StK inhibited bFGF stimulated 10T1/2-MRF4 cell proliferation with dose-dependencies consistent with their FIK blocking actions. ChTX, StK, and IbTX also evoked MRF4-dependent transcription as measured by muscle acetylcholine receptor channel functional expression; but they did not evoke subsequent multinucleated fiber formation or myosin heavy chain expression, suggesting a role for FIK in early, rather than late, myogenic events. Thus despite structural differences, ChTX, IbTX, and StK have common effects on cell growth and differentiation reflecting their common FIK blocking action. We suggest that a major function of the IK channel family is to regulate cell growth.

Cyanide interaction with redox modulatory sites enhances NMDA receptor responses.

Activation of NMDA receptors plays an important role in cyanide neurotoxicity. Cyanide indirectly activates the receptor by inducing neuronal release of glutamate and also enhances receptor-mediated responses by a direct interaction with the receptor complex. This study investigated the mechanism in cerebellar granule cells by which cyanide enhances NMDA-induced Ca2+ influx. Cyanide (50 microM) increased the influx of Ca2+ over the NMDA concentration range of 0.5-500 microM. Experiments showed that cyanide does not interact with the receptor's glycine or PKC mediated phosphorylation regulatory sites. N-ethylmaleimide, a thiol alkylating agent which inactivates the redox regulatory sites of the receptor, blocked the enhancing effect of cyanide. Pretreatment of cells with 5,5-dithio-bis-2-nitrobenzoic acid (DTNB), a compound that oxidizes the receptor redox sites, had no effect on the response to cyanide. On the other hand, the nonpermeant reducing agents, dithiothreitol or cysteine, further increased the cyanide effect. These observations can be explained by cyanide interacting with redox sensitive disulfide groups that are not accessible to the non-permeant reducing agents. It is proposed that cyanide interacts with a redox site(s) located either on the intracellular receptor domain or in the transmembrane hydrophobic domain. Furthermore the enhancement by cyanide of the excitotoxic actions of NMDA involves receptor sites that are sensitive to oxidation/reduction and this interaction contributes to the neurotoxic action of cyanide.

Loss of Cdk4 expression causes insulin-deficient diabetes and Cdk4 activation results in beta-islet cell hyperplasia.

To ascertain the role of cyclin-dependent kinase 4 (Cdk4) in vivo, we have targeted the mouse Cdk4 locus by homologous recombination to generate two strains of mice, one that lacks Cdk4 expression and one that expresses a Cdk4 molecule with an activating mutation. Embryonic fibroblasts proliferate normally in the absence of Cdk4 but have a delayed S phase on re-entry into the cell cycle. Moreover, mice devoid of Cdk4 are viable, but small in size and infertile. These mice also develop insulin-deficient diabetes due to a reduction in beta-islet pancreatic cells. In contrast, mice expressing a mutant Cdk4 that cannot bind the cell-cycle inhibitor P16INK4a display pancreatic hyperplasia due to abnormal proliferation of beta-islet cells. These results establish Cdk4 as an essential regulator of specific cell types.

Differential regulation of the retinoblastoma family of proteins during cell proliferation and differentiation.

In the present study we have analysed the regulation of pocket protein expression and post-transcriptional modifications on cell proliferation and differentiation, both in vivo and in vitro. There are marked changes in pocket protein levels during these transitions, the most striking differences being observed between p130 and p107. The mechanisms responsible for regulating pocket protein levels seem to be dependent on both cell type and pocket protein, in addition to their dependence on the cell growth status. Changes in retinoblastoma protein and p107 levels are independent of their state of phosphorylation. However, whereas p130 phosphorylation to forms characteristic of quiescent/differentiated cells results in the accumulation of p130 protein, phosphorylation of p130 to one or more forms characteristic of cycling cells is accompanied by down-regulation of its protein levels. We also show here that the phosphorylation status and protein levels of p130 and p107 are regulated in vivo as in cultured cells. In vivo, changes in p130 forms are correlated with changes in E2F complexes. Moreover, the modulation of p130 and p107 status during cell differentiation in vitro is consistent with the patterns of protein expression and phosphorylation status found in mouse tissues. Thus in addition to the direct disruption of pocket protein/E2F complexes induced by cyclin/cyclin-dependent kinase, the results we report here indicate that the differential modulation of pocket protein levels constitutes a major mechanism that regulates the pool of each pocket protein that is accessible to E2F and/or other transcription factors.

Growth factor receptor tyrosine kinases acutely regulate neuronal sodium channels through the src signaling pathway.

Growth factor receptor tyrosine kinase (RTK)-activated signaling pathways are well established regulators of neuronal growth and development, but whether these signals provide mechanisms for acute modulation of neuronal activity is just beginning to be addressed. We show in pheochromocytoma (PC12) cells that acute application of ligands for both endogenous RTKs [trkA, basic FGF (bFGF) receptor, and epidermal growth factor (EGF) receptor] and ectopically expressed platelet-derived growth factor (PDGF) receptors rapidly inhibits whole-cell sodium channel currents, coincident with a hyperpolarizing shift in the voltage dependence of inactivation. Sodium channel inhibition by trkA and PDGF receptors is mutually occlusive, suggestive of a common signal transduction mechanism. Furthermore, specific inhibitors for trkA and PDGF RTK activities abrogate sodium channel inhibition in response to NGF and PDGF, respectively, showing that the intrinsic RTK activity of these receptors is necessary for sodium channel inhibition. Use of PDGF receptor mutants deficient for specific signaling activities demonstrated that this inhibition is dependent on RTK interaction with Src but not with other RTK-associated signaling molecules. Inhibition was also compromised in cells expressing dominant-negative Ras. These results suggest a possible mechanism for acute physiological actions of RTKs, and they indicate regulatory functions for Ras and Src that may complement the roles of these signaling proteins in long-term neuronal regulation.

The small conductance calcium-activated potassium channel regulates ion channel expression in C3H10T1/2 cells ectopically expressing the muscle regulatory factor MRF4.

We investigated small conductance (SK) potassium channel-mediated regulation of muscle-specific, ion channel functional expression in the C3H10T1/2-MRF4 cell model system, a stable fibroblast line ectopically overexpressing the myogenic regulatory transcription factor, MRF4. Mitogenic stimulation of C3H10T1/2-MRF4 cells with basic fibroblast growth factor negatively regulates MRF4 transcriptional activity, inhibiting myogenesis. Using patch clamp techniques we found that mitogenic stimulation of C3H10T1/2-MRF4 cells also up-regulated SK. SK is a charybdotoxin-sensitive, apamin-insensitive channel that exerts positive proliferative control in fibroblasts. Mitogen withdrawal, which removes negative regulation of MRF4 thus initiating myogenesis, also eliminated SK channel currents, coincident both with induction of acetylcholine receptor channels, and up-regulation of muscle inward rectifier potassium channels. Addition of the SK channel blocker charybdotoxin to growth factor-containing culture medium overcame basic fibroblast growth factor-induced negative regulation of MRF4, as evidenced by induction of inward rectifier potassium and acetylcholine receptor channel expression identical to that observed in mitogen-withdrawn cells. Thus, the SK channel can govern electrophysiological phenotype in C3H10T1/2-MRF4 cells, consistent with an ability of SK to affect MRF4-dependent transcriptional activity. SK appears to be a pivotal signaling component for growth factor regulation of both cell proliferation and differentiation.

Ca2+-dependent K+ channels in bovine adrenal chromaffin cells are modulated by lipoxygenase metabolites of arachidonic acid.

Fatty acids play an important role in a variety of physiological processes including ion channel modulation and catecholamine release. Using patch-clamp techniques we show that arachidonic acid (AA) is converted to lipoxygenase metabolites (LOMs) to potentiate activity of the Ca2+ and voltage-dependent, large-conductance K+ channel (BK) in bovine adrenal medullary chromaffin cells (BAMCCs). AA and LOM potentiation of BK current and recovery from potentiation were unaffected by the nonhydrolyzable ATP analogue AMP-PNP, or by exclusion of nucleotides in excised patch recordings. Also, AA and LOM potentiation of BK channel activity in outside-out patches exposed to strong Ca2+ buffering ruled out cytoplasmic messengers or changes in intracellular Ca2+ levels as causative factors. Lipoxygenase inhibitor attenuated AA, but not LOM potentiation of BK activity in outside-out patches, indicating that lipoxygenase processing of AA is possible in excised membrane patches, possibly via a membrane associated lipoxygenase. AA and LOM release have been implicated in the mechanics of catecholamine secretion from BAMCCs. By limiting action potential duration and thus voltage-gated Ca2+ influx, fatty acid potentiation of BK current may serve an inhibitory feedback function in regulating secretion from BAMCCs.

Modulation of the NMDA receptor by cyanide: enhancement of receptor-mediated responses.

The effect of cyanide on the N-methyl-D-aspartate (NMDA)-stimulated increase in cytosolic free calcium ([Ca++]i) was studied by microfluorescence in fura-2-loaded cerebellar granule cells. The response to NMDA was enhanced by NaCN over a concentration range of 20 to 100 microM. These concentrations of NaCN in the absence of NMDA had no effect on basal [Ca++]i. In comparison, NaCN did not affect K+-depolarization-induced [Ca++]i elevation. The NaCN potentiation of NMDA-evoked [Ca++]i elevation was blocked by addition of Mg++ and by the NMDA receptor antagonists 2-amino-5-phosphono-valeric acid and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohept-5,10-imine maleate. Pretreatment of the cells with pregnenolone sulfate or arachidonate, known modulators of the NMDA receptor, enhanced NaCN action. The voltage-sensitive calcium channel blockers nifedepine and diltiazem did not affect the NaCN-induced potentiation. Additionally, the NaCN action was not altered when tetrodotoxin was used to block Na+ channel-mediated glutamate release. In patch-clamp studies, NaCN increased the amplitude and duration of NMDA-stimulated whole-cell currents. NaCN also enhanced the NMDA receptor response in single-channel patch-clamp experiments. In the outside-out patch recording configuration, NaCN increased the NMDA receptor channel opening frequency without affecting single-channel conductance or mean channel open time. These results indicate that cyanide interacts directly with the NMDA receptor channel complex to enhance receptor-mediated responses.

EGF in combination with depolarization or cAMP produces morphological but not physiological differentiation in PC12 cells.

In response to nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) receptor activated Ras/extracellular signal-regulated kinase (ERK) signaling, PC12 cells undergo a prototypical neuronal differentiation program, characterized by neurite extension and upregulation of voltage-gated ion channels. The epidermal growth factor (EGF) receptor also activates Ras/ERK signaling, but produces proliferation instead of differentiation. In the presence of depolarizing concentrations of KCl, however, EGF elicits neurite outgrowth through the synergistic actions of the Ras/ERK and cAMP signaling pathways. To assess if EGF and KCl/cAMP elicit the same suite of differentiation events as does NGF and bFGF, we used patch clamp recording to determine if EGF in the presence of KCl or a cAMP agonist also induced physiological differentiation as defined by upregulation of ion channels. Chronic NGF treatment of PC12 cell cultures elicited robust morphological differentiation, a threefold increase in mean calcium channel current density, and an eightfold increase in mean sodium channel current density. Sibling cultures chronically treated with EGF in the presence of high KCl or a cAMP agonist also displayed morphological differentiation, but had calcium channel current densities which were no larger than untreated, undifferentiated cells. Additionally, the increase in mean sodium channel current density induced by EGF in the presence of KCl or cAMP was no greater than the increase observed with EGF alone. Thus, although EGF in the presence of KCl or cAMP is sufficient to induce morphological differentiation as defined by neurite outgrowth, synergism of the Ras/ERK and cAMP/PKA signaling pathways is not sufficient to promote the fully physiologically differentiated PC12 phenotype.

p21ras signaling is necessary but not sufficient to mediate neurotrophin induction of calcium channels in PC12 cells.

Nerve growth factor and basic fibroblast growth factor bind to and activate receptor tyrosine kinases, causing sequential signaling via the p21ras/extracellular signal-regulated kinase pathway. The necessity and sufficiency of this signaling pathway in transducing neuronal differentiation have been tested in the PC12 cell model. Although necessary for morphological changes, the sufficiency of p21ras-mediated signaling in these events has come into question. We report that growth factor induction of voltage-gated calcium channels, a hallmark of physiological differentiation, also requires p21ras-mediated signaling, but cannot be driven by p21ras activation alone. Thus, constitutive expression of the dominant negative N17ras mutant blocks growth factor-induced increases in Omega-conotoxin GVIA-sensitive, nimodipine-sensitive, and Omega-conotoxin GVIA/nimodipine-resistant calcium currents, but it does not block sodium current induction. However, manipulations that produce sustained activation of the p21ras signaling pathway and the neurite extension characteristic of morphological differentiation fail to increase calcium channel current densities. These results indicate the existence of distinct signaling requirements for morphological and physiological differentiation and further emphasize the importance of p21ras-independent signaling pathways in growth factor-mediated neuronal development.