Analysis of ubiquitination and ligand-dependent trafficking of group I mGluRs

Sharma, Rohan; Gulia, Ravinder; Bhattacharyya, Samarjit

doi:10.1016/bs.mcb.2018.08.008

Cited by 4 publications

(5 citation statements)

References 48 publications

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“…The coverslips were then mounted on glass slides and imaged under the confocal microscope. To ensure that the Alexa-647-conjugated secondary antibody did not label any detectable surface receptors in our assays, we performed control experiments to determine the saturating concentration of the first secondary antibody similar to what we have described in our earlier studies (Trivedi and Bhattacharyya, 2012;Pandey et al, 2014;Mahato et al, 2015;Gulia et al, 2017;Sharma et al, 2018Sharma et al, , 2019Pandey et al, 2020). The control experiments suggested that, in our assays, Alexa-647-conjugated secondary antibody did not label any detectable amount of surface receptors; thus, it stained the internalized receptors only (data not shown).…”

Section: Experimental Design and Statistical Analysesmentioning

confidence: 74%

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Regulation of Metabotropic Glutamate Receptor Internalization and Synaptic AMPA Receptor Endocytosis by the Postsynaptic Protein Norbin

2021

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Group I mGluRs have diverse functions in some fundamental neuronal processes, including modulation of synaptic plasticity; and dysregulation of these receptors could lead to various neuropsychiatric disorders. Trafficking of Group I mGluRs plays critical roles in controlling the precise spatiotemporal localization and activity of these receptors, both of which contribute to proper downstream signaling. Using "molecular replacement" approach in hippocampal neurons derived from mice of both sexes, we demonstrate a critical role for the postsynaptic density protein Norbin in regulating the ligand-induced internalization of Group I mGluRs. We show that Norbin associates with protein kinase A (PKA) through its N-terminus and anchors mGluR5 through its C-terminus, both of which are necessary for the ligand-mediated endocytosis of mGluR5, a member of the Group I mGluR family. A point mutation (A687G) at the C-terminus of Norbin inhibits the binding of Norbin to mGluR5 and blocks mGluR5 endocytosis. Finally, we demonstrate an important mechanism by which Norbin regulates mGluR-mediated AMPAR endocytosis in hippocampal neurons, a cellular correlate for mGluR-dependent synaptic plasticity. Norbin, through its PKA-binding regions, recruits PKA to AMPARs on activation of mGluRs; and deletion of the PKA-binding regions of Norbin inhibits mGluR-triggered AMPAR endocytosis. We further report that Norbin is important specifically for the mGluR-mediated AMPAR endocytosis, but not for NMDAR-dependent AMPAR endocytosis. Thus, this study unravels a novel role for Norbin in the internalization of mGluRs and mGluR-mediated AMPAR endocytosis that can have clinical relevance to the function of Group I mGluRs in pathologic processes.

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Section: Experimental Design and Statistical Analysesmentioning

confidence: 74%

“…Primary neurons were transfected with myc-mGluR5/myc-mGluR1, shNor, and various Norbin replacement constructs at 7-8 DIV using calcium phosphate method (Sharma et al, 2018(Sharma et al, , 2019Pandey et al, 2020).…”

Section: Dissociated Primary Neuron Culture and Transfectionmentioning

confidence: 99%

Regulation of Metabotropic Glutamate Receptor Internalization and Synaptic AMPA Receptor Endocytosis by the Postsynaptic Protein Norbin

2021

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“…Neurons were transfected with the indicated fluorescent constructs (0.8 mg DNA for clathrin constructs, 1.2 mg of all others) at 7-9 DIV with either Lipofectamine 2000 (Life Technologies) or Calcium phosphate transfection kit (Promega) (Sharma et al, 2019). Live imaging of axons was performed between 8-10 DIV, and boutons were imaged between 13-21 DIV.…”

Section: Methods Detailsmentioning

confidence: 99%

Clathrin packets move in slow axonal transport and deliver functional payloads to synapses

Ganguly¹,

Sharma²,

Boyer³

et al. 2021

Neuron

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“…Primary hippocampal neurons were transfected with myc-mGluR1/myc-mGluR5, shPICK1, and various PICK1 replacement constructs at 7 to 8 days in vitro (DIV) using calcium phosphate method ( 33 , 44 ). All the experiments were performed when the cells were at 12 to 14 DIV.…”

Section: Methodsmentioning

confidence: 99%

A vital role for PICK1 in the differential regulation of metabotropic glutamate receptor internalization and synaptic AMPA receptor endocytosis

Ramsakha,

Ojha,

Pal

et al. 2023

Journal of Biological Chemistry

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Analysis of ubiquitination and ligand-dependent trafficking of group I mGluRs

Cited by 4 publications

References 48 publications

Regulation of Metabotropic Glutamate Receptor Internalization and Synaptic AMPA Receptor Endocytosis by the Postsynaptic Protein Norbin

Regulation of Metabotropic Glutamate Receptor Internalization and Synaptic AMPA Receptor Endocytosis by the Postsynaptic Protein Norbin

Clathrin packets move in slow axonal transport and deliver functional payloads to synapses

A vital role for PICK1 in the differential regulation of metabotropic glutamate receptor internalization and synaptic AMPA receptor endocytosis

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