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NMDA receptor activation-responsive phosphoproteins in the developing optic tectum
A J Scheetz1, M Constantine-Paton
1Department of Biology, Yale University, New Haven, Connecticut 06520, USA.
Abstract:
A front phosphorylation assay followed by two-dimensional gel electrophoresis was used to detect proteins in the tadpole optic tectum, the phosphorylation state of which is regulated by NMDA receptor activation. Five proteins with isoelectric points between 4 and 7 displayed marked increases in their phosphorylation state in response to application of 10 microM glutamate and 50 microM NMDA. This response was inhibited by 60 microM 2-amino-5-phosphopentanoic acid. These proteins are termed NMDA receptor activation-responsive phosphoproteins (NARPPs). Two NARPPs were identified as both in vitro and in vivo substrates for protein kinase C. Of these two NARPPs, one was located in the postsynaptic density (NARPP-50), and one was located in the nuclear fraction (NARPP-21). Phosphorylation of NARPP-21 was also induced by application of the metabotropic glutamate receptor agonist trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD) (100 microM). Phosphorylation of all NARPPs was eliminated by dantrolene, which inhibits release of calcium from intracellular stores. In adult tecta, only NARPP-21 and -50 were phosphorylation. Thus the phosphorylation state of most NARPPs is regulated differently when synaptic plasticity is low. Further characterization of NARPPs should lead to identification of second messenger systems involved in NMDA receptor signaling and developmental synaptic plasticity.
Insights
Researchers identified novel NMDA receptor activation-responsive phosphoproteins (NARPPs) in tadpole optic tectum. These proteins
Area of Science:
- Neuroscience
- Molecular Biology
- Cellular Signaling
Background:
- NMDA receptor activation plays a crucial role in synaptic plasticity and neuronal development.
- Understanding the downstream signaling pathways of NMDA receptors is essential for elucidating mechanisms of learning and memory.
- The phosphorylation state of proteins is a key regulatory mechanism in cellular processes, including neuronal signaling.
Purpose of the Study:
- To identify and characterize proteins whose phosphorylation is regulated by NMDA receptor activation in the tadpole optic tectum.
- To investigate the role of protein kinase C and intracellular calcium stores in the phosphorylation of these identified proteins.
- To compare the phosphorylation patterns of these proteins in tadpoles versus adult animals to understand developmental changes in synaptic plasticity.
Main Methods:
- Utilized a front phosphorylation assay combined with two-dimensional gel electrophoresis to detect phosphoproteins.
- Applied glutamate, NMDA, and selective receptor agonists/antagonists to modulate NMDA receptor activity.
- Employed dantrolene to assess the involvement of intracellular calcium release.
- Identified specific phosphoproteins using biochemical and subcellular fractionation techniques.
Main Results:
- Five NMDA receptor activation-responsive phosphoproteins (NARPPs) were identified in the tadpole optic tectum.
- Two NARPPs (NARPP-50 and NARPP-21) were confirmed as substrates for protein kinase C, with NARPP-50 localized to the postsynaptic density and NARPP-21 to the nuclear fraction.
- Phosphorylation of NARPP-21 was also induced by a metabotropic glutamate receptor agonist, and phosphorylation of all NARPPs was abolished by dantrolene, indicating calcium dependence.
- In adult tecta, only NARPP-21 and NARPP-50 showed phosphorylation, suggesting altered regulation with decreased synaptic plasticity.
Conclusions:
- The study identified novel NARPPs involved in NMDA receptor signaling pathways.
- These findings highlight the differential regulation of protein phosphorylation during development and its link to synaptic plasticity.
- Further characterization of NARPPs may reveal critical second messenger systems and contribute to understanding developmental synaptic plasticity.