Investigating the mechanism of mrp/plf gene expression by bFGF

The mitogen-regulated protein/proliferin (mrp/ plf) genes belong to the prolactin/growth hormone gene superfamily and encode at least four closely related proteins. Identified functions of these proteins include stimulation of uterine proliferation and endothelial angiogenesis. In 3T3 cells, basic fibroblast growth factor (bFGF) stimulates the production of mrp/plf mRNAs with a resulting increase in the protein products. Although the three cloned mrp/ plf gene promoters (mrp3, plf42, and plf149 ) are over 97% identical in sequence, only mrp3 is transcriptionally activated by bFGF. We have identified a sequence in the mrp3 promoter, which we have named the bFGF-responsive element (FRE), (-186 to -167), that specifically binds nuclear factors from 3T3, CHO and Hela cells. Analysis of the bFGF-responsiveness of a series of truncated mrp3 promoter sequences combined with footprint analysis, pinpointed a region of the promoter that contains a large variation in sequence between the three promoters and one base in the sequence that is unique to the mrp3. The nuclear factors bound by the FRE are present in the placenta and the fetus in which the gene is expressed. By contrast, the maternal liver does not contain FRE-binding proteins. The FRE is transcriptionally active in a TK fusion promoter and responds to bFGF in this context. Our results show that the FRE is an bFGF-responsive transcriptional element. We demonstrate the sequence between -186 to -167 of the three mrp/plf promoters is differentially regulated by bFGF in a TK fusion promoter. Characterization of the 31--36 kDa protein(s) which bind these sequences suggest that different high affinity binding proteins bind to these different sequences. The core sequence of the FRE is also found in the promoters of genes encoding the interstitial collagenase type I and stromelysin-1, that are also regulated by bFGF as delayed early response genes. The FRE element may be the means by which the expression of mrp3 and other genes are regulated by bFG

Investigating the mechanism of mrp/plf gene expression by bFGF

The mitogen-regulated protein/proliferin (mrp/ plf) genes belong to the prolactin/growth hormone gene superfamily and encode at least four closely related proteins. Identified functions of these proteins include stimulation of uterine proliferation and endothelial angiogenesis. In 3T3 cells, basic fibroblast growth factor (bFGF) stimulates the production of mrp/plf mRNAs with a resulting increase in the protein products. Although the three cloned mrp/ plf gene promoters (mrp3, plf42, and plf149 ) are over 97% identical in sequence, only mrp3 is transcriptionally activated by bFGF. We have identified a sequence in the mrp3 promoter, which we have named the "bFGF-responsive element" (FRE), (-186 to -167), that specifically binds nuclear factors from 3T3, CHO and Hela cells. Analysis of the bFGF-responsiveness of a series of truncated mrp3 promoter sequences combined with footprint analysis, pinpointed a region of the promoter that contains a large variation in sequence between the three promoters and one base in the sequence that is unique to the mrp3. The nuclear factors bound by the FRE are present in the placenta and the fetus in which the gene is expressed. By contrast, the maternal liver does not contain FRE-binding proteins. The FRE is transcriptionally active in a TK fusion promoter and responds to bFGF in this context. Our results show that the FRE is an bFGF-responsive transcriptional element. We demonstrate the sequence between -186 to -167 of the three mrp/plf promoters is differentially regulated by bFGF in a TK fusion promoter. Characterization of the 31--36 kDa protein(s) which bind these sequences suggest that different high affinity binding proteins bind to these different sequences. The core sequence of the FRE is also found in the promoters of genes encoding the interstitial collagenase type I and stromelysin-1, that are also regulated by bFGF as delayed early response genes. The FRE element may be the means by which the expression of mrp3 and other genes are regulated by bFGF</p

Coxsackievirus and Adenovirus Receptor (CAR) Mediates Trafficking of Acid Sensing Ion Channel 3 (ASIC3) Via PSD-95

We have previously shown that the Coxsackievirus and adenovirus receptor (CAR) can interact with post-synaptic density 95 (PSD-95) and localize PSD-95 to cell-cell junctions. We have also shown that activity of the acid sensing ion channel (ASIC3), a H+-gated cation channel that plays a role in mechanosensation and pain signaling, is negatively modulated by PSD-95 through a PDZ-based interaction. We asked whether CAR and ASIC3 simultaneously interact with PSD-95, and if so, whether co-expression of these proteins alters their cellular distribution and localization. Results indicate that CAR and ASIC3 co-immunoprecipitate only when co-expressed with PSD-95. CAR also brings both PSD-95 and ASIC3 to the junctions of heterologous cells. Moreover, CAR rescues PSD-95-mediated inhibition of ASIC3 currents. These data suggest that, in addition to activity as a viral receptor and adhesion molecule, CAR can play a role in trafficking proteins, including ion channels, in a PDZ-based scaffolding complex

The Acid-Activated Ion Channel ASIC Contributes to Synaptic Plasticity, Learning, and Memory

AbstractMany central neurons possess large acid-activated currents, yet their molecular identity is unknown. We found that eliminating the acid sensing ion channel (ASIC) abolished H+-gated currents in hippocampal neurons. Neuronal H+-gated currents and transient acidification are proposed to play a role in synaptic transmission. Investigating this possibility, we found ASIC in hippocampus, in synaptosomes, and in dendrites localized at synapses. Moreover, loss of ASIC impaired hippocampal long-term potentiation. ASIC null mice had reduced excitatory postsynaptic potentials and NMDA receptor activation during high-frequency stimulation. Consistent with these findings, null mice displayed defective spatial learning and eyeblink conditioning. These results identify ASIC as a key component of acid-activated currents and implicate these currents in processes underlying synaptic plasticity, learning, and memory

Activation of Acid-sensing Ion Channel 1a (ASIC1a) by Surface Trafficking*

Acid-sensing ion channels (ASICs) are voltage-independent Na+ channels activated by extracellular protons. ASIC1a is expressed in neurons in mammalian brain and is implicated in long term potentiation of synaptic transmission that contributes to learning and memory. In ischemic brain injury, however, activation of this Ca2+-permeable channel plays a critical role in acidosis-mediated, glutamate-independent, Ca2+ toxicity. We report here the identification of insulin as a regulator of ASIC1a surface expression. In modeled ischemia using Chinese hamster ovary cells, serum depletion caused a significant increase in ASIC1a surface expression that resulted in the potentiation of ASIC1a activity. Among the components of serum, insulin was identified as the key factor that maintains a low level of ASIC1a on the plasma membrane. Neurons subjected to insulin depletion increased surface expression of ASIC1a with resultant potentiation of ASIC1a currents. Intracellularly, ASIC1a is predominantly localized to the endoplasmic reticulum in Chinese hamster ovary cells, and this intracellular localization is also observed in neurons. Under conditions of serum or insulin depletion, the intracellular ASIC1a is translocated to the cell surface, increasing the surface expression level. These results reveal an important trafficking mechanism of ASIC1a that is relevant to both the normal physiology and the pathological activity of this channel