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A role for Seven in Absentia Homolog (Siah1a) in metabotropic glutamate receptor signaling.

Kammermeier PJ, Ikeda SR - BMC Neurosci (2001)

Bottom Line: The effects of coexpression of Siah on group I mGluR signaling were examined using heterologous expression in rat sympathetic, superior cervical ganglion neurons.Finally, coexpression of calmodulin, which competes with Siah1a for binding to the C-terminal tail of group I mGluRs, reversed the effect of Siah1a on mGluR-mediated signaling.These data supported the conclusion that the attenuation of mGluR signaling induced by Siah1a expression was likely a direct consequence of Siah/mGluR association rather than a result of targeting of the receptors to the proteosome.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Molecular Physiology, Guthrie Research Institute, Sayre, PA 18840, USA. pkammerm@inet.guthrie.org

ABSTRACT

Background: The mammalian homologue of Seven in Absentia (Siah) can act in the ubiquitin/proteasome pathway. Recent work has shown that Siah can bind group I metabotropic glutamate receptors (mGluRs), but the functional consequences of this interaction are unknown.

Results: The effects of coexpression of Siah on group I mGluR signaling were examined using heterologous expression in rat sympathetic, superior cervical ganglion neurons. Siah1a attenuated heterologously expressed group I mGluR-mediated calcium current inhibition, but was without effect on group II mGluR- or NE-mediated calcium current modulation via heterologously expressed mGluR2 or native a2 adrenergic receptors, respectively, indicating that the effect of Siah was specific for group I mGluRs. Surface expression and subcellular distribution of group I mGluRs were not detectably altered in the presence of Siah1a as assessed by immunoflourescence experiments with epitope tagged receptors and imaging of a GFP/mGluR fusion construct. In addition, an N-terminal Siah deletion construct, which cannot function in the proteolysis pathway, displayed effects similar to the wild type Siah1a. Finally, coexpression of calmodulin, which competes with Siah1a for binding to the C-terminal tail of group I mGluRs, reversed the effect of Siah1a on mGluR-mediated signaling.

Conclusions: These data supported the conclusion that the attenuation of mGluR signaling induced by Siah1a expression was likely a direct consequence of Siah/mGluR association rather than a result of targeting of the receptors to the proteosome. In addition, the data suggest that the binding of CaM and Siah may play an important role in the regulation of group I mGluR function.

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Effect of Siah1a expression on mGluR-mediated calcium current modulation. A and C, Sample current traces illustrating calcium current before and during inhibition by 100 μM glutamate in cells expressing mGluR5b (A) and mGluR5b + Siah1a (C). A triple pulse voltage protocol was used wherein cells were sustained at a holding potential of-80 mV and stepped to an initial test pulse to +10 mV for 25 msec, to +80 mV for 50 msec, then following a 10 msec step to -80 mV a second test pulse to +10 mV was applied. The scale bars indicate 20 msec and 0.4 nA. Tail currents were cropped to better illustrate the step currents. B and D, Time course of inhibition by glutamate (Glu) and norepinephrine (NE) in the cells illustrated in A and C, respectively. Closed circles represent measurements taken from the prepulse (the first test pulse to +10 mV). Open circles represent measurements taken from the postpulse (the second test pulse to +10 mV).
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Figure 1: Effect of Siah1a expression on mGluR-mediated calcium current modulation. A and C, Sample current traces illustrating calcium current before and during inhibition by 100 μM glutamate in cells expressing mGluR5b (A) and mGluR5b + Siah1a (C). A triple pulse voltage protocol was used wherein cells were sustained at a holding potential of-80 mV and stepped to an initial test pulse to +10 mV for 25 msec, to +80 mV for 50 msec, then following a 10 msec step to -80 mV a second test pulse to +10 mV was applied. The scale bars indicate 20 msec and 0.4 nA. Tail currents were cropped to better illustrate the step currents. B and D, Time course of inhibition by glutamate (Glu) and norepinephrine (NE) in the cells illustrated in A and C, respectively. Closed circles represent measurements taken from the prepulse (the first test pulse to +10 mV). Open circles represent measurements taken from the postpulse (the second test pulse to +10 mV).

Mentions: To examine the role of Siah1a expression on mGluR function, SCG neurons were injected with cDNA encoding several group I mGluRs (mGluR1, 5 and their splice variants) with or without Siah1a expression. Calcium current modulation was then examined upon application of extracellular 100 μM glutamate. The triple pulse voltage protocol [39] was used as the standard test pulse to assess calcium current magnitude and voltage dependence of inhibition [38,40]. Figure 1A illustrates sample current traces from before ("con") and during inhibition by glutamate ("Glu"), in an mGluR5b-expressing cell. Consistent with previous reports [24,38], calcium current inhibition via mGluR5b appeared partially voltage dependent. The time course of inhibition by glutamate and 10 μM norepinephrine (NE), acting through endogenous α2 adrenergic receptors [41], in this cell (figure 1B) illustrates that inhibition was rapid and reversible.


A role for Seven in Absentia Homolog (Siah1a) in metabotropic glutamate receptor signaling.

Kammermeier PJ, Ikeda SR - BMC Neurosci (2001)

Effect of Siah1a expression on mGluR-mediated calcium current modulation. A and C, Sample current traces illustrating calcium current before and during inhibition by 100 μM glutamate in cells expressing mGluR5b (A) and mGluR5b + Siah1a (C). A triple pulse voltage protocol was used wherein cells were sustained at a holding potential of-80 mV and stepped to an initial test pulse to +10 mV for 25 msec, to +80 mV for 50 msec, then following a 10 msec step to -80 mV a second test pulse to +10 mV was applied. The scale bars indicate 20 msec and 0.4 nA. Tail currents were cropped to better illustrate the step currents. B and D, Time course of inhibition by glutamate (Glu) and norepinephrine (NE) in the cells illustrated in A and C, respectively. Closed circles represent measurements taken from the prepulse (the first test pulse to +10 mV). Open circles represent measurements taken from the postpulse (the second test pulse to +10 mV).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC58838&req=5

Figure 1: Effect of Siah1a expression on mGluR-mediated calcium current modulation. A and C, Sample current traces illustrating calcium current before and during inhibition by 100 μM glutamate in cells expressing mGluR5b (A) and mGluR5b + Siah1a (C). A triple pulse voltage protocol was used wherein cells were sustained at a holding potential of-80 mV and stepped to an initial test pulse to +10 mV for 25 msec, to +80 mV for 50 msec, then following a 10 msec step to -80 mV a second test pulse to +10 mV was applied. The scale bars indicate 20 msec and 0.4 nA. Tail currents were cropped to better illustrate the step currents. B and D, Time course of inhibition by glutamate (Glu) and norepinephrine (NE) in the cells illustrated in A and C, respectively. Closed circles represent measurements taken from the prepulse (the first test pulse to +10 mV). Open circles represent measurements taken from the postpulse (the second test pulse to +10 mV).
Mentions: To examine the role of Siah1a expression on mGluR function, SCG neurons were injected with cDNA encoding several group I mGluRs (mGluR1, 5 and their splice variants) with or without Siah1a expression. Calcium current modulation was then examined upon application of extracellular 100 μM glutamate. The triple pulse voltage protocol [39] was used as the standard test pulse to assess calcium current magnitude and voltage dependence of inhibition [38,40]. Figure 1A illustrates sample current traces from before ("con") and during inhibition by glutamate ("Glu"), in an mGluR5b-expressing cell. Consistent with previous reports [24,38], calcium current inhibition via mGluR5b appeared partially voltage dependent. The time course of inhibition by glutamate and 10 μM norepinephrine (NE), acting through endogenous α2 adrenergic receptors [41], in this cell (figure 1B) illustrates that inhibition was rapid and reversible.

Bottom Line: The effects of coexpression of Siah on group I mGluR signaling were examined using heterologous expression in rat sympathetic, superior cervical ganglion neurons.Finally, coexpression of calmodulin, which competes with Siah1a for binding to the C-terminal tail of group I mGluRs, reversed the effect of Siah1a on mGluR-mediated signaling.These data supported the conclusion that the attenuation of mGluR signaling induced by Siah1a expression was likely a direct consequence of Siah/mGluR association rather than a result of targeting of the receptors to the proteosome.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Molecular Physiology, Guthrie Research Institute, Sayre, PA 18840, USA. pkammerm@inet.guthrie.org

ABSTRACT

Background: The mammalian homologue of Seven in Absentia (Siah) can act in the ubiquitin/proteasome pathway. Recent work has shown that Siah can bind group I metabotropic glutamate receptors (mGluRs), but the functional consequences of this interaction are unknown.

Results: The effects of coexpression of Siah on group I mGluR signaling were examined using heterologous expression in rat sympathetic, superior cervical ganglion neurons. Siah1a attenuated heterologously expressed group I mGluR-mediated calcium current inhibition, but was without effect on group II mGluR- or NE-mediated calcium current modulation via heterologously expressed mGluR2 or native a2 adrenergic receptors, respectively, indicating that the effect of Siah was specific for group I mGluRs. Surface expression and subcellular distribution of group I mGluRs were not detectably altered in the presence of Siah1a as assessed by immunoflourescence experiments with epitope tagged receptors and imaging of a GFP/mGluR fusion construct. In addition, an N-terminal Siah deletion construct, which cannot function in the proteolysis pathway, displayed effects similar to the wild type Siah1a. Finally, coexpression of calmodulin, which competes with Siah1a for binding to the C-terminal tail of group I mGluRs, reversed the effect of Siah1a on mGluR-mediated signaling.

Conclusions: These data supported the conclusion that the attenuation of mGluR signaling induced by Siah1a expression was likely a direct consequence of Siah/mGluR association rather than a result of targeting of the receptors to the proteosome. In addition, the data suggest that the binding of CaM and Siah may play an important role in the regulation of group I mGluR function.

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