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Mutation p.R356Q in the Collybistin Phosphoinositide Binding Site Is Associated With Mild Intellectual Disability

Tzu-Ting Chiou, Philip Long, Alexandra Schumann-Gillett, Venkat Kanamarlapudi Orcid Logo, Stefan A. Haas, Kirsten Harvey, Megan L. O’Mara, Angel L. De Blas, Vera M. Kalscheuer, Robert J. Harvey

Frontiers in Molecular Neuroscience, Volume: 12

Swansea University Author: Venkat Kanamarlapudi Orcid Logo

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DOI (Published version): 10.3389/fnmol.2019.00060

Abstract

The recruitment of inhibitory GABAA receptors to neuronal synapses requires a complex interplay between receptors, neuroligins, the scaffolding protein gephyrin and the GDP-GTP exchange factor collybistin (CB). Collybistin is regulated by protein-protein interactions at the N-terminal SH3 domain, wh...

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Published in: Frontiers in Molecular Neuroscience
ISSN: 1662-5099
Published: Frontiers Media SA 2019
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URI: https://cronfa.swan.ac.uk/Record/cronfa50064
first_indexed 2019-04-26T13:37:57Z
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Collybistin also harbors a RhoGEF domain which mediates interactions with gephyrin and catalyzes GDP-GTP exchange on Cdc42. Lastly, collybistin has a pleckstrin homology (PH) domain, which binds phosphoinositides, such as phosphatidylinositol 3-phosphate (PI3P/PtdIns3P) and phosphatidylinositol 4-monophosphate (PI4P/PtdIns4P). PI3P located in early/sorting endosomes has recently been shown to regulate the postsynaptic clustering of gephyrin and GABAA receptors and consequently the strength of inhibitory synapses in cultured hippocampal neurons. This process is disrupted by mutations in the collybistin gene (ARHGEF9), which cause X-linked intellectual disability (XLID) by a variety of mechanisms converging on disrupted gephyrin and GABAA receptor clustering at central synapses. Here we report a novel missense mutation (chrX:62875607C&gt;T, p.R356Q) in ARHGEF9 that affects one of the two paired arginine residues in the PH domain that were predicted to be vital for binding phosphoinositides. Functional assays revealed that recombinant collybistin CB3SH3-R356Q was deficient in PI3P binding and was not able to translocate EGFP-gephyrin to submembrane microaggregates in an in vitro clustering assay. Expression of the PI3P-binding mutants CB3SH3-R356Q and CB3SH3-R356N/R357N in cultured hippocampal neurones revealed that the mutant proteins did not accumulate at inhibitory synapses, but instead resulted in a clear decrease in the overall number of synaptic gephyrin clusters compared to controls. Molecular dynamics simulations suggest that the p.R356Q substitution influences PI3P binding by altering the range of structural conformations adopted by collybistin. Taken together, these results suggest that the p.R356Q mutation in ARHGEF9 is the underlying cause of XLID in the probands, disrupting gephyrin clustering at inhibitory GABAergic synapses via loss of collybistin PH domain phosphoinositide binding.</abstract><type>Journal Article</type><journal>Frontiers in Molecular Neuroscience</journal><volume>12</volume><journalNumber/><paginationStart/><paginationEnd/><publisher>Frontiers Media SA</publisher><placeOfPublication/><isbnPrint/><isbnElectronic/><issnPrint/><issnElectronic>1662-5099</issnElectronic><keywords>ARHGEF9, Collybistin, Gephyrin, PH domain, PI3P, XLID</keywords><publishedDay>12</publishedDay><publishedMonth>3</publishedMonth><publishedYear>2019</publishedYear><publishedDate>2019-03-12</publishedDate><doi>10.3389/fnmol.2019.00060</doi><url/><notes/><college>COLLEGE NANME</college><department>Medical School</department><CollegeCode>COLLEGE CODE</CollegeCode><DepartmentCode>MEDS</DepartmentCode><institution>Swansea University</institution><apcterm/><lastEdited>2021-11-19T03:58:58.3987663</lastEdited><Created>2019-04-23T15:36:30.0620651</Created><path><level id="1">Faculty of Medicine, Health and Life Sciences</level><level id="2">Swansea University Medical School - Medicine</level></path><authors><author><firstname>Tzu-Ting</firstname><surname>Chiou</surname><order>1</order></author><author><firstname>Philip</firstname><surname>Long</surname><order>2</order></author><author><firstname>Alexandra</firstname><surname>Schumann-Gillett</surname><order>3</order></author><author><firstname>Venkat</firstname><surname>Kanamarlapudi</surname><orcid>0000-0002-8739-1483</orcid><order>4</order></author><author><firstname>Stefan A.</firstname><surname>Haas</surname><order>5</order></author><author><firstname>Kirsten</firstname><surname>Harvey</surname><order>6</order></author><author><firstname>Megan L.</firstname><surname>O&#x2019;Mara</surname><order>7</order></author><author><firstname>Angel L. 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spelling 2021-11-19T03:58:58.3987663 v2 50064 2019-04-23 Mutation p.R356Q in the Collybistin Phosphoinositide Binding Site Is Associated With Mild Intellectual Disability 63741801137148abfa4c00cd547dcdfa 0000-0002-8739-1483 Venkat Kanamarlapudi Venkat Kanamarlapudi true false 2019-04-23 MEDS The recruitment of inhibitory GABAA receptors to neuronal synapses requires a complex interplay between receptors, neuroligins, the scaffolding protein gephyrin and the GDP-GTP exchange factor collybistin (CB). Collybistin is regulated by protein-protein interactions at the N-terminal SH3 domain, which can bind neuroligins 2/4 and the GABAAR a2 subunit. Collybistin also harbors a RhoGEF domain which mediates interactions with gephyrin and catalyzes GDP-GTP exchange on Cdc42. Lastly, collybistin has a pleckstrin homology (PH) domain, which binds phosphoinositides, such as phosphatidylinositol 3-phosphate (PI3P/PtdIns3P) and phosphatidylinositol 4-monophosphate (PI4P/PtdIns4P). PI3P located in early/sorting endosomes has recently been shown to regulate the postsynaptic clustering of gephyrin and GABAA receptors and consequently the strength of inhibitory synapses in cultured hippocampal neurons. This process is disrupted by mutations in the collybistin gene (ARHGEF9), which cause X-linked intellectual disability (XLID) by a variety of mechanisms converging on disrupted gephyrin and GABAA receptor clustering at central synapses. Here we report a novel missense mutation (chrX:62875607C>T, p.R356Q) in ARHGEF9 that affects one of the two paired arginine residues in the PH domain that were predicted to be vital for binding phosphoinositides. Functional assays revealed that recombinant collybistin CB3SH3-R356Q was deficient in PI3P binding and was not able to translocate EGFP-gephyrin to submembrane microaggregates in an in vitro clustering assay. Expression of the PI3P-binding mutants CB3SH3-R356Q and CB3SH3-R356N/R357N in cultured hippocampal neurones revealed that the mutant proteins did not accumulate at inhibitory synapses, but instead resulted in a clear decrease in the overall number of synaptic gephyrin clusters compared to controls. Molecular dynamics simulations suggest that the p.R356Q substitution influences PI3P binding by altering the range of structural conformations adopted by collybistin. Taken together, these results suggest that the p.R356Q mutation in ARHGEF9 is the underlying cause of XLID in the probands, disrupting gephyrin clustering at inhibitory GABAergic synapses via loss of collybistin PH domain phosphoinositide binding. Journal Article Frontiers in Molecular Neuroscience 12 Frontiers Media SA 1662-5099 ARHGEF9, Collybistin, Gephyrin, PH domain, PI3P, XLID 12 3 2019 2019-03-12 10.3389/fnmol.2019.00060 COLLEGE NANME Medical School COLLEGE CODE MEDS Swansea University 2021-11-19T03:58:58.3987663 2019-04-23T15:36:30.0620651 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Medicine Tzu-Ting Chiou 1 Philip Long 2 Alexandra Schumann-Gillett 3 Venkat Kanamarlapudi 0000-0002-8739-1483 4 Stefan A. Haas 5 Kirsten Harvey 6 Megan L. O’Mara 7 Angel L. De Blas 8 Vera M. Kalscheuer 9 Robert J. Harvey 10 0050064-23042019154126.pdf CollybistinR356QPIbindinginteldisabilityFIMN19.pdf 2019-04-23T15:41:26.7400000 Output 7312733 application/pdf Version of Record true Released under the terms of a Creative Commons Attribution License (CC-BY). true eng http://creativecommons.org/licenses/by/4.0/
title Mutation p.R356Q in the Collybistin Phosphoinositide Binding Site Is Associated With Mild Intellectual Disability
spellingShingle Mutation p.R356Q in the Collybistin Phosphoinositide Binding Site Is Associated With Mild Intellectual Disability
Venkat Kanamarlapudi
title_short Mutation p.R356Q in the Collybistin Phosphoinositide Binding Site Is Associated With Mild Intellectual Disability
title_full Mutation p.R356Q in the Collybistin Phosphoinositide Binding Site Is Associated With Mild Intellectual Disability
title_fullStr Mutation p.R356Q in the Collybistin Phosphoinositide Binding Site Is Associated With Mild Intellectual Disability
title_full_unstemmed Mutation p.R356Q in the Collybistin Phosphoinositide Binding Site Is Associated With Mild Intellectual Disability
title_sort Mutation p.R356Q in the Collybistin Phosphoinositide Binding Site Is Associated With Mild Intellectual Disability
author_id_str_mv 63741801137148abfa4c00cd547dcdfa
author_id_fullname_str_mv 63741801137148abfa4c00cd547dcdfa_***_Venkat Kanamarlapudi
author Venkat Kanamarlapudi
author2 Tzu-Ting Chiou
Philip Long
Alexandra Schumann-Gillett
Venkat Kanamarlapudi
Stefan A. Haas
Kirsten Harvey
Megan L. O’Mara
Angel L. De Blas
Vera M. Kalscheuer
Robert J. Harvey
format Journal article
container_title Frontiers in Molecular Neuroscience
container_volume 12
publishDate 2019
institution Swansea University
issn 1662-5099
doi_str_mv 10.3389/fnmol.2019.00060
publisher Frontiers Media SA
college_str Faculty of Medicine, Health and Life Sciences
hierarchytype
hierarchy_top_id facultyofmedicinehealthandlifesciences
hierarchy_top_title Faculty of Medicine, Health and Life Sciences
hierarchy_parent_id facultyofmedicinehealthandlifesciences
hierarchy_parent_title Faculty of Medicine, Health and Life Sciences
department_str Swansea University Medical School - Medicine{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Medicine
document_store_str 1
active_str 0
description The recruitment of inhibitory GABAA receptors to neuronal synapses requires a complex interplay between receptors, neuroligins, the scaffolding protein gephyrin and the GDP-GTP exchange factor collybistin (CB). Collybistin is regulated by protein-protein interactions at the N-terminal SH3 domain, which can bind neuroligins 2/4 and the GABAAR a2 subunit. Collybistin also harbors a RhoGEF domain which mediates interactions with gephyrin and catalyzes GDP-GTP exchange on Cdc42. Lastly, collybistin has a pleckstrin homology (PH) domain, which binds phosphoinositides, such as phosphatidylinositol 3-phosphate (PI3P/PtdIns3P) and phosphatidylinositol 4-monophosphate (PI4P/PtdIns4P). PI3P located in early/sorting endosomes has recently been shown to regulate the postsynaptic clustering of gephyrin and GABAA receptors and consequently the strength of inhibitory synapses in cultured hippocampal neurons. This process is disrupted by mutations in the collybistin gene (ARHGEF9), which cause X-linked intellectual disability (XLID) by a variety of mechanisms converging on disrupted gephyrin and GABAA receptor clustering at central synapses. Here we report a novel missense mutation (chrX:62875607C>T, p.R356Q) in ARHGEF9 that affects one of the two paired arginine residues in the PH domain that were predicted to be vital for binding phosphoinositides. Functional assays revealed that recombinant collybistin CB3SH3-R356Q was deficient in PI3P binding and was not able to translocate EGFP-gephyrin to submembrane microaggregates in an in vitro clustering assay. Expression of the PI3P-binding mutants CB3SH3-R356Q and CB3SH3-R356N/R357N in cultured hippocampal neurones revealed that the mutant proteins did not accumulate at inhibitory synapses, but instead resulted in a clear decrease in the overall number of synaptic gephyrin clusters compared to controls. Molecular dynamics simulations suggest that the p.R356Q substitution influences PI3P binding by altering the range of structural conformations adopted by collybistin. Taken together, these results suggest that the p.R356Q mutation in ARHGEF9 is the underlying cause of XLID in the probands, disrupting gephyrin clustering at inhibitory GABAergic synapses via loss of collybistin PH domain phosphoinositide binding.
published_date 2019-03-12T08:30:08Z
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