Tracking the estrogen receptor in neurons: Implications for estrogen-induced synapse formation

Estrogens (E) and progestins regulate synaptogenesis in the CA1 region of the dorsal hippocampus during the estrous cycle of the female rat, and the functional consequences include changes in neurotransmission and memory. Synapse formation has been demonstrated by using the Golgi technique, dye filling of cells, electron microscopy, and radioimmunocytochemistry. N-methyl-d-aspartate (NMDA) receptor activation is required, and inhibitory interneurons play a pivotal role as they express nuclear estrogen receptor alpha (ERα) and show E-induced decreases of GABAergic activity. Although global decreases in inhibitory tone may be important, a more local role for E in CA1 neurons seems likely. The rat hippocampus expresses both ERα and ERβ mRNA. At the light microscopic level, autoradiography shows cell nuclear [3H]estrogen and [125I]estrogen uptake according to a distribution that primarily reflects the localization of ERα-immunoreactive interneurons in the hippocampus. However, recent ultrastructural studies have revealed extranuclear ERα immunoreactivity (IR) within select dendritic spines on hippocampal principal cells, axon terminals, and glial processes, localizations that would not be detectable by using standard light microscopic methods. Based on recent studies showing that both types of ER are expressed in a form that activates second messenger systems, these findings support a testable model in which local, non-genomic regulation by estrogen participates along with genomic actions of estrogens in the regulation of synapse formation.

A High-Affinity Ca2+ Pump, ECA1, from the Endoplasmic Reticulum Is Inhibited by Cyclopiazonic Acid but Not by Thapsigargin1

To identify and characterize individual Ca2+ pumps, we have expressed an Arabidopsis ECA1 gene encoding an endoplasmic reticulum-type Ca2+-ATPase homolog in the yeast (Saccharomyces cerevisiae) mutant K616. The mutant (pmc1pmr1cnb1) lacks a Golgi and a vacuolar membrane Ca2+ pump and grows very poorly on Ca2+-depleted medium. Membranes isolated from the mutant showed high H+/Ca2+-antiport but no Ca2+-pump activity. Expression of ECA1 in endomembranes increased mutant growth by 10- to 20-fold in Ca2+-depleted medium. 45Ca2+ pumping into vesicles from ECA1 transformants was detected after the H+/Ca2+-antiport activity was eliminated with bafilomycin A1 and gramicidin D. The pump had a high affinity for Ca2+ (Km = 30 nm) and displayed two affinities for ATP (Km of 20 and 235 μm). Cyclopiazonic acid, a specific blocker of animal sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, inhibited Ca2+ transport (50% inhibition dose = 3 nmol/mg protein), but thapsigargin (3 μm) did not. Transport was insensitive to calmodulin. These results suggest that this endoplasmic reticulum-type Ca2+-ATPase could support cell growth in plants as in yeast by maintaining submicromolar levels of cytosolic Ca2+ and replenishing Ca2+ in endomembrane compartments. This study demonstrates that the yeast K616 mutant provides a powerful expression system to study the structure/function relationships of Ca2+ pumps from eukaryotes.

Transport of Sterols to the Plasma Membrane of Leek Seedlings1

To investigate the intracellular transport of sterols in etiolated leek (Allium porrum L.) seedlings, in vivo pulse-chase experiments with [1-14C]acetate were performed. Then, endoplasmic reticulum-, Golgi-, and plasma membrane (PM)-enriched fractions were prepared and analyzed for the radioactivity incorporated into free sterols. In leek seedlings sterols are present as a mixture in which (24R)-24-ethylcholest-5-en-3β-ol is by far the major compound (around 60%). The other sterols are represented by cholest-5-en-3β-ol, 24-methyl-cholest-5-en-3β-ol, (24S)-24-ethylcholesta-5,22E-dien-3β-ol, and stigmasta-5,24(241)Z-dien-3β-ol. These compounds are shown to reside mainly in the PM. Our results clearly indicate that free sterols are actively transported from the endoplasmic reticulum to the PM during the first 60 min of chase, with kinetics very similar to that of phosphatidylserine. Such a transport was found to be decreased at low temperature (12°C) and following treatment with monensin and brefeldin A. These data are consistent with a membrane-mediated process for the intracellular transport of sterols to the PM, which likely involves the Golgi apparatus.

Transdifferentiation of Mature Cortical Cells to Functional Abscission Cells in Bean1

Abscission explants of bean (Phaseolus vulgaris L.) were treated with ethylene to induce cell separation at the primary abscission zone. After several days of further incubation of the remaining petiole in endogenously produced ethylene, the distal two-thirds of the petiole became senescent, and the remaining (proximal) portion stayed green. Cell-to-cell separation (secondary abscission) takes place precisely at the interface between the senescing yellow and the enlarging green cells. The expression of the abscission-associated isoform of β-1,4-glucanhydrolase, the activation of the Golgi apparatus, and enhanced vesicle formation occurred only in the enlarging cortical cells on the green side. These changes were indistinguishable from those that occur in normal abscission cells and confirm the conversion of the cortical cells to abscission-type cells. Secondary abscission cells were also induced by applying auxin to the exposed primary abscission surface after the pulvinus was shed, provided ethylene was added. Then, the orientation of development of green and yellow tissue was reversed; the distal tissue remained green and the proximal tissue yellowed. Nevertheless, separation still occurred at the junction between green and yellow cells and, again, it was one to two cell layers of the green side that enlarged and separated from their senescing neighbors. Evaluation of Feulgen-stained tissue establishes that, although nuclear changes occur, the conversion of the cortical cell to an abscission zone cell is a true transdifferentiation event, occurring in the absence of cell division.

Ras Regulates the Polarity of the Yeast Actin Cytoskeleton through the Stress Response Pathway

Polarized growth in yeast requires cooperation between the polarized actin cytoskeleton and delivery of post-Golgi secretory vesicles. We have previously reported that loss of the major tropomyosin isoform, Tpm1p, results in cells sensitive to perturbations in cell polarity. To identify components that bridge these processes, we sought mutations with both a conditional defect in secretion and a partial defect in polarity. Thus, we set up a genetic screen for mutations that conferred a conditional growth defect, showed synthetic lethality with tpm1Δ, and simultaneously became denser at the restrictive temperature, a hallmark of secretion-defective cells. Of the 10 complementation groups recovered, the group with the largest number of independent isolates was functionally null alleles of RAS2. Consistent with this, ras2Δ and tpm1Δ are synthetically lethal at 35°C. We show that ras2Δ confers temperature-sensitive growth and temperature-dependent depolarization of the actin cytoskeleton. Furthermore, we show that at elevated temperatures ras2Δ cells are partially defective in endocytosis and show a delocalization of two key polarity markers, Myo2p and Cdc42p. However, the conditional enhanced density phenotype of ras2Δ cells is not a defect in secretion. All the phenotypes of ras2Δ cells can be fully suppressed by expression of yeast RAS1 or RAS2 genes, human Ha-ras, or the double disruption of the stress response genes msn2Δmsn4Δ. Although the best characterized pathway of Ras function in yeast involves activation of the cAMP-dependent protein kinase A pathway, activation of the protein kinase A pathway does not fully suppress the actin polarity defects, suggesting that there is an additional pathway from Ras2p to Msn2/4p. Thus, Ras2p regulates cytoskeletal polarity in yeast under conditions of mild temperature stress through the stress response pathway.

A Novel Quality Control Compartment Derived from the Endoplasmic Reticulum

Degradation of proteins that, because of improper or suboptimal processing, are retained in the endoplasmic reticulum (ER) involves retrotranslocation to reach the cytosolic ubiquitin-proteasome machinery. We found that substrates of this pathway, the precursor of human asialoglycoprotein receptor H2a and free heavy chains of murine class I major histocompatibility complex (MHC), accumulate in a novel preGolgi compartment that is adjacent to but not overlapping with the centrosome, the Golgi complex, and the ER-to-Golgi intermediate compartment (ERGIC). On its way to degradation, H2a associated increasingly after synthesis with the ER translocon Sec61. Nevertheless, it remained in the secretory pathway upon proteasomal inhibition, suggesting that its retrotranslocation must be tightly coupled to the degradation process. In the presence of proteasomal inhibitors, the ER chaperones calreticulin and calnexin, but not BiP, PDI, or glycoprotein glucosyltransferase, concentrate in the subcellular region of the novel compartment. The “quality control” compartment is possibly a subcompartment of the ER. It depends on microtubules but is insensitive to brefeldin A. We discuss the possibility that it is also the site for concentration and retrotranslocation of proteins that, like the mutant cystic fibrosis transmembrane conductance regulator, are transported to the cytosol, where they form large aggregates, the “aggresomes.”

Thapsigargin-induced transport of cholera toxin to the endoplasmic reticulum.

Cholera toxin is normally observed only in the Golgi apparatus and not in the endoplasmic reticulum (ER) although the enzymatically active A subunit of cholera toxin has a KDEL sequence. Here we demonstrate transport of horseradish peroxidase-labeled cholera toxin to the ER by electron microscopy in thapsigargin-treated A431 cells. Thapsigargin treatment strongly increased cholera toxin-induced cAMP production, and the formation of the catalytically active A1 fragment was somewhat increased. Binding of cholera toxin to the cell surface and transport of toxin to the Golgi apparatus were not changed in thapsigargin-treated cells, suggesting increased retrograde transport of cholera toxin from the Golgi apparatus to the ER. The data demonstrate that retrograde transport of cholera toxin can take place and that the transport is under regulation. The results are consistent with the idea that retrograde transport can be important for the action of cholera toxin.

Ethanol causes translocation of cAMP-dependent protein kinase catalytic subunit to the nucleus.

Short- and long-term ethanol exposures have been shown to alter cellular levels of cAMP, but little is known about the effects of ethanol on cAMP-dependent protein kinase (PKA). When cAMP levels increase, the catalytic subunit of PKA (C alpha) is released from the regulatory subunit, phosphorylates nearby proteins, and then translocates to the nucleus, where it regulates gene expression. Altered localization of C alpha would have profound effects on multiple cellular functions. Therefore, we investigated whether ethanol alters intracellular localization of C alpha. NG108-15 cells were incubated in the presence or absence of ethanol for as long as 48 h, and localization of PKA subunits was determined by immunocytochemistry. We found that ethanol exposure produced a significant translocation of C alpha from the Golgi area to the nucleus. C alpha remained in the nucleus as long as ethanol was present. There was no effect of ethanol on localization of the type I regulatory subunit of PKA. Ethanol also caused a 43% decrease in the amount of type I regulatory subunit but had no effect on the amount of C alpha as determined by Western blot. These data suggest that ethanol-induced translocation of C alpha to the nucleus may account, in part, for diverse changes in cellular function and gene expression produced by alcohol.

Effects of inefficient cleavage of the signal sequence of HIV-1 gp 120 on its association with calnexin, folding, and intracellular transport.

The HIV-1 envelope glycoprotein gp120 displays inefficient intracellular transport, which is caused by its retention in the endoplasmic reticulum. Coexpression in insect cells (Sf9) of HIV-1 gp120 with calnexin has shown that their interaction was modulated by the signal sequence of HIV-1 gp120. gp120, with its natural signal sequence, showed a prolonged association with calnexin with a t1/2 of greater than 20 min. Replacement of the natural signal sequence with the signal sequence from mellitin led to a decreased time of association of gp120 with calnexin (t1/2 < 10 min). These different times of calnexin association coincided both with the folding of gp120 as measured by the ability of bind CD4 and with endoplasmic reticulum to Golgi transport as analyzed by the acquisition of partial endoglycosidase H resistance. Using a monospecific antibody to the HIV-1 gp120 natural signal peptide, we showed that calnexin associated with N-glycosylated but uncleaved gp120. Only after dissociation from calnexin was gp120 cleaved, but very inefficiently. Only the small proportion of signal-cleaved gp120 molecules acquired transport competence and were secreted. This is the first report demonstrating the effect of the signal sequence on calnexin association.

Interleaflet clear space is reduced in the membrane of COP I and COP II-coated buds/vesicles.

Intracellular transfers between membrane-bound compartments occur through vesicles that bud from a donor compartment to fuse subsequently with an acceptor membrane. We report that the membrane that delimits COP I or COP II-coated buds/vesicles from the endoplasmic reticulum and the Golgi complex has a thinner interleaflet clear space as compared with the surrounding, noncoated parental membrane. This change is compatible with a compositional change of the membrane bilayer during the budding process.

Real-time detection of the surface delivery of newly synthesized membrane proteins.

Newly synthesized membrane proteins travel from the Golgi complex to the cell surface in transport vesicles. We have exploited the ion channel properties of the nicotinic acetylcholine receptor (AChR) to observe in real time the constitutive delivery of newly synthesized AChR proteins to the plasma membrane in cultured muscle cells. Whole-cell voltage clamp was employed to monitor the current fluctuations induced by carbamylcholine upon the insertion into the plasma membrane of newly synthesized AChRs, following release from a 20 degrees C temperature block. We find that the transit of vesicles to the cell surface occurs within a few minutes after release of the block. The time course of electrical signals is consistent with many of the fusion events being instantaneous, although some appear to reveal the flickering of a fusion pore. AChR-containing vesicles can fuse individually or as conglomerates. Intracellular application of guanosine 5'-[gamma-thio]triphosphate inhibits the constitutive traffic of AChRs in most cells. Individual exocytotic vesicles carry between 10 and 300 AChR molecules, suggesting that AChRs may be packed extremely densely.

Cholinergic agonists stimulate secretion of soluble full-length amyloid precursor protein in neuroendocrine cells.

The Abeta peptide of Alzheimer disease is derived from the proteolytic processing of the amyloid precursor proteins (APP), which are considered type I transmembrane glycoproteins. Recently, however, soluble forms of full-length APP were also detected in several systems including chromaffin granules. In this report we used antisera specific for the cytoplasmic sequence of APP to show that primary bovine chromaffin cells secrete a soluble APP, termed solAPPcyt, of an apparent molecular mass of 130 kDa. This APP was oversecreted from Chinese hamster ovary cells transfected with a full-length APP cDNA indicating that solAPPcyt contained both the transmembrane and Abeta sequence. Deglycosylation of solAPPcyt showed that it contained both N- and O-linked sugars, suggesting that this APP was transported through the endoplasmic reticulum-Golgi pathway. Secretion of solAPPcyt from primary chromatin cells was temperature-, time-, and energy-dependent and was stimulated by cell depolarization in a Ca2+-dependent manner. Cholinergic receptor agonists, including acetylcholine, nicotine, or carbachol, stimulated the rapid secretion of solAPPcyt, a process that was inhibited by cholinergic antagonists. Stimulation of solAPPcyt secretion was paralleled by a stimulation of secretion in catecholamines and chromogranin A, indicating that secretion of solAPPcyt was mediated by chromaffin granule vesicles. Taken together, our results show that release of the potentially amyloidogenic solAPPcyt is an active cellular process mediated by both the constitutive and regulated pathways. solAPPcyt was also detected in human cerebrospinal fluid. Combined with the neuronal physiology of chromaffin cells, our data suggest that cholinergic agonists may stimulate the release of this APP in neuronal synapses where it may exert its biological functions. Moreover, vesicular or secreted solAPPcyt may serve as a soluble precursor of Abeta.

Targeting of nitric oxide synthase to endothelial cell caveolae via palmitoylation: implications for nitric oxide signaling.

The membrane association of endothelial nitric oxide synthase (eNOS) plays an important role in the biosynthesis of nitric oxide (NO) in vascular endothelium. Previously, we have shown that in cultured endothelial cells and in intact blood vessels, eNOS is found primarily in the perinuclear region of the cells and in discrete regions of the plasma membrane, suggesting trafficking of the protein from the Golgi to specialized plasma membrane structures. Here, we show that eNOS is found in Triton X-100-insoluble membranes prepared from cultured bovine aortic endothelial cells and colocalizes with caveolin, a coat protein of caveolae, in cultured bovine lung microvascular endothelial cells as determined by confocal microscopy. To examine if eNOS is indeed in caveolae, we purified luminal endothelial cell plasma membranes and their caveolae directly from intact, perfused rat lungs. eNOS is found in the luminal plasma membranes and is markedly enriched in the purified caveolae. Because palmitoylation of eNOS does not significantly influence its membrane association, we next examined whether this modification can affect eNOS targeting to caveolae. Wild-type eNOS, but not the palmitoylation mutant form of the enzyme, colocalizes with caveolin on the cell surface in transfected NIH 3T3 cells, demonstrating that palmitoylation of eNOS is necessary for its targeting into caveolae. These data suggest that the subcellular targeting of eNOS to caveolae can restrict NO signaling to specific targets within a limited microenvironment at the cell surface and may influence signal transduction through caveolae.

In its active form, the GTP-binding protein rab8 interacts with a stress-activated protein kinase.

Rab8 is a small GTP-binding protein that plays a role in vesicular transport from the trans-Golgi network to the basolateral plasma membrane in polarized epithelial cells (MDCK), and to the dendritic surface in hippocampal neurons. As is the case for most other rab proteins, the precise molecular interactions by which rab8 carries out its function remain to be elucidated. Here we report the identification and the complete cDNA-derived amino acid sequence of a murine rab8-interacting protein (rab8ip) that specifically interacts with rab8 in a GTP-dependent manner. Rab8ip displays 93% identity with the GC kinase, a serine/threonine protein kinase recently identified in human lymphoid tissue that is activated in the stress response. Like the GC kinase, rab8ip has protein kinase activity manifested by autophosphorylation and phosphorylation of the classical serine/threonine protein kinase substrates, myelin basic protein and casein. When coexpressed in transfected 293T cells, rab8 and the rab8ip/GC kinase formed a complex that could be recovered by immunoprecipitation with antibodies to rab8. Cell fractionation and immunofluorescence analyses indicate that in MDCK cells endogenous rab8ip is present both in the cytosol and as a peripheral membrane protein concentrated in the Golgi region and basolateral plasma membrane domains, sites where rab8 itself is also located. In light of recent evidence that rab proteins may act by promoting the stabilization of SNARE complexes, the specific GTP-dependent association of rab8 with the rab8ip/GC kinase raises the possibility that rab-regulated protein phosphorylation is important for vesicle targeting or fusion. Moreover, the rab8ip/GC kinase may serve to modulate secretion in response to stress stimuli.

Metabolism of Alzheimer beta-amyloid precursor protein: regulation by protein kinase A in intact cells and in a cell-free system.

Various compounds that affect signal transduction regulate the relative utilization of alternative processing pathways for the beta-amyloid precursor protein (beta APP) in intact cells, increasing the production of nonamyloidogenic soluble beta APP (s beta APP) and decreasing that of amyloidogenic beta-amyloid peptide. In a recent study directed toward elucidating the mechanisms underlying phorbol ester-stimulated s beta APP secretion from cells, it was demonstrated that protein kinase C increases the formation from the trans-Golgi network (TGN) of beta APP-containing secretory vesicles. Here we present evidence that forskolin increases s beta APP production from intact PC12 cells, and protein kinase A stimulates formation from the TGN of beta APP-containing vesicles. Although protein kinase A and protein kinase C converge at the level of formation from the TGN of beta APP-containing vesicles, additional evidence indicates that the regulatory mechanisms involved are distinct.