Biomedical subjects

D M Fass

Publications and source records attributed to D M Fass.

9 recordsLinked to original sources

PubMed2000

Cooperative mechanism of transcriptional activation by a cyclic AMP-response element modulator alpha mutant containing a motif for constitutive binding to CREB-binding protein.

Cyclic AMP-response element modulator alpha (CREMalpha) is a transcription factor that is highly related to cAMP-response element-binding protein (CREB) but represses cAMP-induced gene expression from simple artificial promoters containing a cAMP-response element (CRE). CREMalpha lacks two glutamine-rich Q regions that, in CREB, are thought to be necessary for transcriptional activation. Nevertheless, protein kinase A stimulation induces CREMalpha to activate the complex native promoter in the phosphoenolpyruvate carboxykinase (PEPCK) gene. To study this phenomenon in the absence of protein kinase A stimulation, we introduced a mutation into CREMalpha to allow constitutive binding to the coactivator CREB-binding protein. This mutant, CREMalpha(DIEDML), constitutively activated the PEPCK promoter. By engineering the leucine zipper regions of CREMalpha(DIEDML) and CREB(DIEDML) to direct their patterns of dimerization, we found that only CREMalpha(DIEDML) homodimers fully activated the PEPCK promoter. By using a series of deletion and block mutants of the PEPCK promoter, we found that activation by CREMalpha(DIEDML) depended on the CRE and two CCAAT/enhancer-binding protein (C/EBP) sites. A dominant negative inhibitor of C/EBP, A-C/EBP, suppressed activation by CREMalpha(DIEDML). Furthermore, a GAL4-C/EBPalpha fusion protein and CREMalpha(DIEDML) cooperatively activated a promoter containing three GAL4 sites and the PEPCK CRE. Thus, we propose that the C/EBP sites in the PEPCK promoter allow CREMalpha to activate transcription despite its lack of Q regions.

Amino Acid Motifs↗

PubMed2000

Calcium and cAMP signals differentially regulate cAMP-responsive element-binding protein function via a Rap1-extracellular signal-regulated kinase pathway.

Two major intracellular signals that regulate neuronal function are calcium and cAMP. In many cases, the actions of these two second messengers involve long term changes in gene expression. One well studied target of both calcium and cAMP signaling is the transcription factor cAMP-responsive element-binding protein (CREB). Multiple signaling pathways have been shown to contribute to the regulation of CREB-dependent transcription, including both protein kinase A (PKA)- and mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK)-dependent kinase cascades. We have previously described a mechanism by which cAMP and calcium influx may stimulate ERKs in neuronal cells. This pathway involves the PKA-dependent activation of the Ras-related small G-protein, Rap1, and subsequent stimulation of the neuronal Raf isoform, B-Raf. In this study, we examined the contribution of the Rap1-ERK pathway to the control of gene transcription by calcium influx and cAMP. Using the PC12 cell model system, we found that both calcium influx and cAMP stimulated CREB-dependent transcription via a Rap1-ERK pathway, but this regulation occurred through distinct mechanisms. Calcium-mediated phosphorylation of CREB through the PKA-Rap1-ERK pathway. In contrast, cAMP phosphorylated CREB via PKA directly but required a Rap1-ERK pathway to activate a component downstream of CREB phosphorylation and CREB-binding protein recruitment. These data suggest that the Rap1/B-Raf signaling pathway may have an important role in the regulation of CREB-dependent gene expression.

3T3 Cells↗

PubMed2000

Recruitment of CREB binding protein is sufficient for CREB-mediated gene activation.

Phosphorylation of the transcription factor CREB leads to the recruitment of the coactivator, CREB binding protein (CBP). Recent studies have suggested that CBP recruitment is not sufficient for CREB function, however. We have identified a conserved protein-protein interaction motif within the CBP-binding domains of CREB and another transcription factor, SREBP (sterol-responsive element binding protein). In contrast to CREB, SREBP interacts with CBP in the absence of phosphorylation. We have exploited the conservation of this interaction motif to test whether CBP recruitment to CREB is sufficient for transcriptional activation. Substitution of six nonconserved amino acids from SREBP into the activation domain of CREB confers high-affinity, phosphorylation-independent CBP binding. The mutated CREB molecule, CREB(DIEDML), activates transcription in F9 teratocarcinoma and PC12 cells even in the absence of protein kinase A (PKA). Addition of exogenous CBP augments the level of transcription mediated by CREB(DIEDML), and adenovirus 12S E1A blocks transcription, implicating CBP in the activation process. Thus, recruitment of CBP to CREB is sufficient for transcriptional activation. Addition of PKA stimulates transcription induced by CREB(DIEDML) further, suggesting that a phosphorylation event downstream from CBP recruitment augments CREB signaling.

Amino Acid Sequence↗

PubMed1999

Emergence of excitotoxicity in cultured forebrain neurons coincides with larger glutamate-stimulated [Ca(2+)](i) increases and NMDA receptor mRNA levels.

We examined several factors related to the increase in susceptibility to excitotoxicity that occurs in embryonic forebrain neurons over time in culture. Neuronal cultures were resistant to a 5-min exposure to 100 microM glutamate/10 microM glycine at 5 days in vitro (DIV), but became vulnerable to the same stimulus by 14 DIV. We used the fluorescent indicators, fura-2 and magfura-2, which have high and low affinity for Ca(2+), respectively, to measure changes in [Ca(2+)](i). Glutamate-stimulated increases in the fura-2 and magfura-2 ratio reached maximum values by 10 DIV. Fura-2 reported similar [Ca(2+)](i) changes with exposure to 3 or 100 microM glutamate for 5 min, whereas magfura-2 reported larger [Ca(2+)](i) increases with 5-min exposure to 100 microM glutamate than with exposure to 3 microM glutamate, 100 microM kainate or 50 mM K(+) from 10 DIV onward. This suggests that the magnitude of the [Ca(2+)](i) changes correlated with the excitotoxicity potential of a stimulus when magfura-2, but not fura-2, was used to measure Ca(2+). We also used RNase protection assays to measure NMDA receptor subunit mRNA levels. NR1 and NR2A mRNA increased continuously over time in culture, whereas NR2B mRNA increased dramatically during the first 10 days and subsequently remained stable. The time course of the increase in NR2B mRNA most closely followed the increase in glutamate-stimulated changes in the magfura-2 signal and neuronal injury. Therefore, the increases in the glutamate-stimulated [Ca(2+)](i) responses and NMDA receptor subunit mRNA levels (especially NR2B) are likely involved in the development of susceptibility to excitotoxicity in cultured rat forebrain neurons.

Animals↗

PubMed1999

Tonic dopamine inhibition of L-type Ca2+ channel activity reduces alpha1D Ca2+ channel gene expression.

Hormones and neurotransmitters have both short-term and long-term modulatory effects on the activity of voltage-gated Ca2+ channels. Although much is known about the signal transduction underlying short-term modulation, there is far less information on mechanisms that produce long-term effects. Here, the molecular basis of long-lasting suppression of Ca2+ channel current in pituitary melanotropes by chronic dopamine exposure is examined. Experiments involving in vivo and in vitro treatments with the dopaminergic drugs haloperidol, bromocriptine, and quinpirole show that D2 receptors persistently decrease alpha1D L-type Ca2+ channel mRNA and L-type Ca2+ channel current without altering channel gating properties. In contrast, another L-channel (alpha1C) mRNA and P/Q-channel (alpha1A) mRNA are unaffected. The downregulation of alpha1D mRNA does not require decreases in cAMP levels or P/Q-channel activity. However, it is mimicked and occluded by inhibition of L-type channels. Thus, interruption of the positive feedback between L-type Ca2+ channel activity and alpha1D gene expression can account for the long-lasting regulation of L-current produced by chronic activation of D2 dopamine receptors.

Animals↗

PubMed1996

Bay K 8644 reveals two components of L-type Ca2+ channel current in clonal rat pituitary cells.

Whole-cell L-type Ca2+ channel current was recorded in GH3 clonal rat pituitary cells using Ba2+ as a charge carrier. In the presence of the dihydropyridine agonist Bay K 8644, deactivation was best described by two exponential components with time constants of approximately 2 and approximately 8 ms when recorded at -40 mV. The slow component activated at more negative potentials than the fast component: Half-maximal activation for the slow and fast components occurred at approximately -15 and approximately 1 mV, respectively. The fast component was more sensitive to enhancement by racemic Bay K 8644 than the slow component: ED50fast = approximately 21 nM, ED50slow = approximately 74 nM. Thyrotropin-releasing hormone (TRH; 1 microM) inhibited the slow component by approximately 46%, whereas the fast component was inhibited by approximately 22%. TRH inhibition of total L-current showed some voltage dependence, but each Bay K 8644-revealed component of L-current was inhibited in a voltage-independent manner. Therefore, the apparent voltage dependence of TRH action is derived from complexities in channel gating rather than from relief of inhibition at high voltages. In summary, Bay K 8644-enhanced L-currents in GH3 cells consist of two components with different sensitivities to voltage, racemic Bay K 8644, and the neuropeptide TRH.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1996

L-type Ca2+ channels access multiple open states to produce two components of Bay K 8644-dependent current in GH3 cells.

To determine the number of L-channel populations responsible for producing the two components of whole-cell L-type Ca2+ channel current revealed by Bay K 8644 (Fass, D.M., and E.S. Levitan. 1996. J. Gen. Physiol. 108:1-11), L-type Ca2+ channel activity was recorded in cell-attached patches. Ensemble tail currents from most (six out of nine) single-channel patches had double-exponential time courses, with time constants that were similar to whole-cell tail current decay values. Also, in single-channel patches subjected to two different levels of depolarization, ensemble tail currents exactly reproduced the voltage dependence of activation of the two whole-cell components: The slow component is activated at more negative potentials than the fast component. In addition, deactivation of Bay K 8644-modified whole-cell L-current was slower after long (100-ms) depolarizations than after short (20-ms) depolarizations, and this phenomenon was also evident in ensemble tail currents from single L-channels. Thus, a single population of L-channels can produce the two components of macroscopic L-current deactivation. To determine how individual L-channels produce multiple macroscopic tail current components, we constructed ensemble tail currents from traces that contained a single opening upon repolarization and no reopenings. These ensemble tails were biexponential. This type of analysis also revealed that reopenings do not contribute to the slowing of tail current deactivation after long depolarizations. Thus, individual L-channels must have access to several open states to produce multiple macroscopic current components. We also obtained evidence that access to these open states can vary over time. Use of several open states may give L-channels the flexibility to participate in many cell functions.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1994

Current, voltage and pharmacological substrates of a novel GABA receptor in the visual-vestibular system of Hermissenda.

In the marine mollusc, Hermissenda crassicornis, Type B photoreceptors exhibit an IPSP to both presynaptic hair cell stimulation and microapplication of gamma-aminobutyric acid (GABA) to the terminal branches. It was found that both the endogenous IPSP and the response to exogenously applied GABA were mediated to a large part by an outward current which reversed at approximately -80 mV. Additionally, these hyperpolarizing responses were found to mask a smaller depolarization that was mediated by the reduction of a basal outward current. Both the IPSP and the hyperpolarizing response to GABA, as well as the sublimated depolarizing response to GABA, were attenuated by the K+ channel blocker tetraethylammonium chloride (TEA) and displayed a strong sensitivity to [K+]o, while showing no sensitivity to [Cl-]o or the Cl- channel blocker picrotoxin. Moreover, iontophoretic injections of stable guanine analogues, GTP[gamma S] and GDP[beta S], into B photoreceptors eliminated both the IPSP and the GABA-induced hyperpolarization, while cholinergically mediated, interphotoreceptor interactions were unaffected. These results suggest that the endogenous receptor is at least partially homologous to the mammalian GABAB class receptor. Consistent with this classification, microapplication of selective GABAB receptor agonist baclofen onto the terminal region of the B photoreceptor resulted in a hyperpolarizing response that was qualitatively similar to that of GABA, although the GABAA agonist muscimol was also active, but less so than either GABA or baclofen. Attempts to block the endogenous IPSP or GABA-induced hyperpolarization by bath application of the GABAA receptor subtype antagonist bicuculline was ineffective and the GABAB receptor subtype antagonist saclofen was only weakly effective. These data demonstrate that the presynaptic hair cell's influence on postsynaptic B photoreceptors is in many respects similar to GABAB mediated responses in the mammalian CNS. This receptor is in some respects unique, however, in terms of its cross-sensitivity to both GABAA and GABAB agonists, its weak sensitivity to saclofen, and its apparent anomalous modulation of multiple K+ conductances.

Animals↗

PubMed1993

GABA-mediated synaptic interaction between the visual and vestibular pathways of Hermissenda.

The synaptic convergence of the eyes and the vestibular hair cells in the nudibranch mollusc Hermissenda has been shown previously to mediate the learning of simple visual-vestibular associations. The neurotransmitter mediating this interaction between the visual and vestibular organs was characterized. HPLC chromatography, confirmed by mass spectroscopic analysis, demonstrated endogenous GABA in the statocysts, in a concentration approximately 150 times greater than in the whole CNS. Additional confirmation was provided by immunocytochemical localization of GABA in hair cell axons and branches that converge with photoreceptor terminal branches. Depolarization of the hair cells in the caudal region of the statocyst in response to positive current injection or vibratory stimulation caused a hyperpolarization and a cessation of the type B photoreceptor impulse activity. The inhibition of the B cell was unaffected by addition to the artificial sea water bath of the adrenergic antagonist yohimbine (250 microM), the cholinergic antagonist atropine (250 microM), and the serotonergic antagonist imipramine (50 microM). In contrast, the GABAA antagonist bicuculline (250 microM) significantly reduced the inhibitory interaction. Moreover, the GABA reuptake inhibitor guvisine (250 microM) increased the hyperpolarization. Pressure microapplication of GABA (12.5 or 25 microM) onto the terminal branches of the B cell resulted in a concentration-dependent hyperpolarization and cessation of spikes in the B cell. Depolarization of the caudal hair cell, or direct GABA application, decreased input resistance across the B cell soma membrane. Moreover, removal of chloride from the extracellular solution reduced inhibition of the B cell induced by GABA application or hair cell stimulation. Furthermore, application of the GABAB agonist baclofen hyperpolarized the type B cell and reduced or eliminated spontaneous impulse activity at the resting membrane potential. The reversal potentials for inhibition induced in all three procedures ranged from -70 to -80 mV and were consistent with mixed Cl- and K+ conductances. These results implicate GABA as the endogenous neurotransmitter mediating visual-vestibular interactions in this animal, and suggest a possible role of GABA in visual-vestibular associative learning.

Animals↗