Milestone Review: Unlocking the Proteomics of Glycine Receptor Complexes

“The BioID approach first utilized an E. coli biotin ligase mutant (BirA*), which generates reactive biotin (biotinoyl- 5-AMP) and has an enhanced off-rate, meaning that biotin covalently attaches to exposed lysine residues of any neighboring protein within 10 nm (Roux et al. 2012; Guo et al. 2023).”

“…on synaptic PPIs is integrated into publicly available platforms such as SynGO (Synaptic Gene Ontology), an expert-curated universal reference for synapse research and an online analysis platform for interpretation of large-scale proteomics data (Koopmans et al. 2019; https:// syngo portal. org).”

“Molecular cloning revealed that the 48 kDa and 58 kDa components represent the GlyR α1 and β subunits (Grenningloh et al. 1987; Grenningloh, Pribilla et al. 1990), while the 93 kDa component is a synaptic scaffolding protein known as gephyrin (from the Greek geph-yra , for bridge; Prior et al.…”

“…β subunits is the TM3-TM4 intracellular domain (ICD), which provides sites for interactions with accessory proteins and subunit-specific modulation by ubiquitination (Büttner et al. 2001) or phosphorylation by protein kinases (Ruiz-Gómez et al. 1991; Harvey, Depner et al. 2004; Manzke et al. 2010).”

“Hence, GlyR-importin interactions may be ar-tifactual as isolated ICDs may show false-positive interactions in the yeast two-hybrid system (Bruckner et al. 2009) or mis-localization to cellular compartments that are not representative of the behavior of full-length GlyR subunits (Melzer et al.…”

“This autoinhibition can be relieved by the binding of several neuronal interactors to the collybistin SH3 domain, including neuroligins 2 and 4 (Poulopoulos et al. 2009; Hoon et al. 2011), the GABA A R α2 subunit (Saiepour et al. 2010) or the binding of the small GTPase TC10 to the PH domain (Imam…”

“Accordingly, GlyR α1β dysfunction causes startle disease/hyperekplexia, characterized by exaggerated startle reflexes in response to unexpected stimuli, muscle hypertonia, and neonatal apnea episodes (Shiang et al. 1993; Rees et al. 2002; Schaefer et al. 2022).”

“The latter is consistent with a region-specific loss of clustering of some, but not all, GABA A R subtypes in gephyrin-deficient mice (Kneussel et al. 1999, 2001; Fischer et al. 2000; Lévi et al. 2004), but it means that gephyrin is a key synaptic clustering protein at both glycinergic and…”

“Gephyrin knockdown using antisense oligonucleotides in spinal cord neurons or in knockout mice prevents GlyR clustering (Kirsch et al. 1993; Feng et al. 1998; Fischer et al. 2000).”

“Gephyrin knockdown using antisense oligonucleotides in spinal cord neurons or in knockout mice prevents GlyR clustering (Kirsch et al. 1993; Feng et al. 1998; Fischer et al. 2000).”

“Each GlyR subunit comprises an N-terminal signal peptide (SP), an agonist-binding extracellular domain (ECD), four transmembrane domains (TM1-TM4) an intracellular domain (ICD) between TM3-TM4, and a short extracellular C-terminus (Du et al. 2015; Yu et al. 2021; Zhu and Gouaux 2021).”

“The GlyR β subunit binds to the gephyrin E domain via the short motif 398 FSIVGSLPRDFELS 411 in the TM3-TM4 ICD (Figure 2; Kirsch et al. 1995; Meyer et al. 1995; Rees et al. 2003; Harvey, Duguid et al. 2004; Schrader et al. 2004; Sola et al. 2004; Kim et al. 2006).”

“Initially affinity-purified from the rat and pig spinal cord in the early 1980s (Pfeiffer et al. 1982; Graham et al. 1985) by amino strychnine affinity chromatography, GlyR preparations contained three major proteins of molecular weights 48 kDa, 58 kDa, and 93 kDa.”

“Following injection of these fusion protein constructs encased in an adeno-associated virus (AAV) vector and exogenous biotin supplementation, biotinylated proteins were affinity-purified by streptavidin and identified by MS (Uezu et al. 2016).”

“Gephyrin is perhaps best known for its role in synaptic clustering of GlyRs, but it also has a key role in the synthesis of molybdenum cofactor (Moco) required for the activity of enzymes sulfite oxidase, xanthine oxidase, and aldehyde oxidase (Feng et al. 1998; Fritschy et al. 2008).”

“Both homopentameric α or heteromeric αβ receptors can be formed in vitro, which differ in pharmacology and ion-channel conductance states (Pribilla et al. 1992; Bormann et al. 1993).”

“0; Liu et al. 2017).”

“…microfilament components such as G-actin, profilin, Mena/ VASP (Giesemann et al. 2003), dynein light chains 1 and 2 (Fuhrmann et al. 2002), tubulin (Kirsch et al. 1991) and several other synaptic proteins including the RhoGEF collybis-tin (Kins et al. 2000; Grosskreutz et al. 2001; Harvey, Duguid…”

“…inhibitory synapses, and artificial or X-linked intellectual disability/epilepsy mutations affecting the SH3 or PH domains cause mis-localization of geph-yrin and inhibitory receptors (Kins et al. 2000; Harvey, Duguid et al. 2004; Kalscheuer et al. 2009; Reddy-Alla et al. 2010; Chiou et al. 2019).”

“The GlyR β subunit binds to the gephyrin E domain via the short motif 398 FSIVGSLPRDFELS 411 in the TM3-TM4 ICD (Figure 2; Kirsch et al. 1995; Meyer et al. 1995; Rees et al. 2003; Harvey, Duguid et al. 2004; Schrader et al. 2004; Sola et al. 2004; Kim et al. 2006).”

“…neurobeachin (Lutzenkirchen et al. 2024), neuroligins 2 and 4 (Poulopoulos et al. 2009; Hoon et al. 2011) and importantly the TM3-TM4 ICDs of GABA A R α1, α2, α3 and α5 subunits (Tretter et al. 2008, 2011; Saiepour et al. 2010; Mukherjee et al. 2011; Maric et al. 2011, 2014; Brady and Jacob 2015).”

“…consistent with a region-specific loss of clustering of some, but not all, GABA A R subtypes in gephyrin-deficient mice (Kneussel et al. 1999, 2001; Fischer et al. 2000; Lévi et al. 2004), but it means that gephyrin is a key synaptic clustering protein at both glycinergic and GABAergic synapses .”

“Molecular cloning revealed that the 48 kDa and 58 kDa components represent the GlyR α1 and β subunits (Grenningloh et al. 1987; Grenningloh, Pribilla et al. 1990), while the 93 kDa component is a synaptic scaffolding protein known as gephyrin (from the Greek geph-yra , for bridge; Prior et al.…”

“…can be relieved by the binding of several neuronal interactors to the collybistin SH3 domain, including neuroligins 2 and 4 (Poulopoulos et al. 2009; Hoon et al. 2011), the GABA A R α2 subunit (Saiepour et al. 2010) or the binding of the small GTPase TC10 to the PH domain (Imam et al. 2022).”

“This may be consistent with previous studies suggesting that collybistin is not required for GlyR synaptic localization (Papadopoulos et al. 2007).”

“The GlyR α1-α4 subunits exhibit a high degree of sequence identity ( ≥ 80%; Grenningloh, Schmieden, et al. 1990; Kuhse et al. 1990; Matzenbach et al. 1994) which hampered the design of subunit-specific antibodies for many years.”

“However, it is noteworthy that the ECDs of neurexins can bind directly to the GABA A R α1 subunit ECD in a Ca 2+ dependent manner (Zhang et al. 2010).”

“The latter is consistent with a region-specific loss of clustering of some, but not all, GABA A R subtypes in gephyrin-deficient mice (Kneussel et al. 1999, 2001; Fischer et al. 2000; Lévi et al. 2004), but it means that gephyrin is a key synaptic clustering protein at both glycinergic and…”

“Gephyrin forms multimers via a trimeric N-terminal G domain, a central alternatively spliced C domain, and dimeric C-terminal E domain (Prior et al. 1992; Rees et al. 2003; Harvey, Duguid et al. 2004; Sola et al. 2004).”

“…neurobeachin (Lutzenkirchen et al. 2024), neuroligins 2 and 4 (Poulopoulos et al. 2009; Hoon et al. 2011) and importantly the TM3-TM4 ICDs of GABA A R α1, α2, α3 and α5 subunits (Tretter et al. 2008, 2011; Saiepour et al. 2010; Mukherjee et al. 2011; Maric et al. 2011, 2014; Brady and Jacob 2015).”

“In addition, VPS35 localizes to dendritic spines and influences the trafficking of excitatory AMPARs (Munsie et al. 2015; Temkin et al. 2017).”

“Both homopentameric α or heteromeric αβ receptors can be formed in vitro, which differ in pharmacology and ion-channel conductance states (Pribilla et al. 1992; Bormann et al. 1993).”

“The GlyR β subunit binds to the gephyrin E domain via the short motif 398 FSIVGSLPRDFELS 411 in the TM3-TM4 ICD (Figure 2; Kirsch et al. 1995; Meyer et al. 1995; Rees et al. 2003; Harvey, Duguid et al. 2004; Schrader et al. 2004; Sola et al. 2004; Kim et al. 2006).”

“…inhibitory synapses, and artificial or X-linked intellectual disability/epilepsy mutations affecting the SH3 or PH domains cause mis-localization of geph-yrin and inhibitory receptors (Kins et al. 2000; Harvey, Duguid et al. 2004; Kalscheuer et al. 2009; Reddy-Alla et al. 2010; Chiou et al. 2019).”

“…can be relieved by the binding of several neuronal interactors to the collybistin SH3 domain, including neuroligins 2 and 4 (Poulopoulos et al. 2009; Hoon et al. 2011), the GABA A R α2 subunit (Saiepour et al. 2010) or the binding of the small GTPase TC10 to the PH domain (Imam et al. 2022).”

“The GlyR α1-α4 subunits exhibit a high degree of sequence identity ( ≥ 80%; Grenningloh, Schmieden, et al. 1990; Kuhse et al. 1990; Matzenbach et al. 1994) which hampered the design of subunit-specific antibodies for many years.”

“These auxiliary proteins include the lipoma HMGIC fusion partner-like 3 and 4 (LHFPL3/LHFPL4; Davenport et al. 2017; Yamasaki et al. 2017 Han et al. 2019) or (iv) promoting cell-surface expression while also slowing GABA A R deactivation (TMEM132B; Wang et al. 2024).”

“Initially affinity-purified from the rat and pig spinal cord in the early 1980s (Pfeiffer et al. 1982; Graham et al. 1985) by amino strychnine affinity chromatography, GlyR preparations contained three major proteins of molecular weights 48 kDa, 58 kDa, and 93 kDa.”

“Second, gephyrin is a highly promiscuous protein , interacting with microfilament components such as G-actin, profilin, Mena/ VASP (Giesemann et al. 2003), dynein light chains 1 and 2 (Fuhrmann et al. 2002), tubulin (Kirsch et al. 1991) and several other synaptic proteins including the RhoGEF…”

“Gephyrin forms multimers via a trimeric N-terminal G domain, a central alternatively spliced C domain, and dimeric C-terminal E domain (Prior et al. 1992; Rees et al. 2003; Harvey, Duguid et al. 2004; Sola et al. 2004).”

“Notably, spontaneous GlyR activity has also been identified as a potential cause of startle disease for other autosomal-dominant inherited GlyR α1 mutations (Chung et al. 2010; Bode et al. 2013).”

“…in a range of neuro-developmental disorders (NDDs) featuring autism spectrum disorder (ASD), developmental delay (DD), intellectual disability (ID) often accompanied by microcephaly, language delay, and epilepsy (Pilorge et al. 2016; Zhang et al. 2017; Chen et al. 2022; Marcogliese et al. 2022).”

“One possibility in this regard is the use of cross-linking MS (XL-MS; Liu and Heck 2015; O'Reilly and Rappsilber 2018), a method that involves the use of a soluble, MS-cleavable cross-linker such as disuccinimidyl sulfoxide (DSSO), followed by the downstream analysis of cross-linked peptides via MS…”

“Mutagenesis studies revealed that hydrophobic interactions formed by F330, Y673, and P713 of gephyrin and F398 and I400 of the GlyR β ICD are critical for binding (Kim et al. 2006; Maric et al. 2011).”

“They obtained 11999 unique cross-links corresponding to connections within and between 2362 proteins and demonstrated high fidelity and reproducibility as an experimental approach (Gonzalez-Lozano et al. 2020).”

“Additionally, true direct interactors can fail to be affinity purified due to factors such as improper folding or lack of post-translational modification of bait proteins (Mackay et al. 2007).”

“These auxiliary proteins include the lipoma HMGIC fusion partner-like 3 and 4 (LHFPL3/LHFPL4; Davenport et al. 2017; Yamasaki et al. 2017 Han et al. 2019) or (iv) promoting cell-surface expression while also slowing GABA A R deactivation (TMEM132B; Wang et al. 2024).”

“The SH3 domain regulates the gephyrin-clustering activity of collybistin (Kins et al. 2000; Harvey, Duguid et al. 2004) via autoinhibition of PH domain phosphoinositide-binding and gephyrin-clustering activity (Soykan et al. 2014).”

“First, gephyrin is a large protein that binds to GlyR β with high affinity (Schrader et al. 2004; Kim et al. 2006; Specht et al. 2011) meaning that: (i) gephyrin peptides are over-represented in mass spectrometry (MS) analysis of GlyR IPs or GST pulldowns and (ii) other lower-affinity GlyR β…”

“The development of GlyR α2 knockout mice established a major role for GlyR α2 in cortical interneuron migration (Avila et al. 2013, 2014).”

“First, gephyrin is a large protein that binds to GlyR β with high affinity (Schrader et al. 2004; Kim et al. 2006; Specht et al. 2011) meaning that: (i) gephyrin peptides are over-represented in mass spectrometry (MS) analysis of GlyR IPs or GST pulldowns and (ii) other lower-affinity GlyR β…”

“Neurobeachin interacts with Dlg3 (SAP102), a member of the membrane-associated guanylate kinase protein family, and is required for synaptic surface expression of AMPARs, NMDARs, and GABA A Rs (Nair et al. 2013; Farzana et al. 2016).”

“These results suggest that the role of the GlyR α3L-SEC8 interaction is to facilitate axonal trafficking of GlyR α3L-SEC8 complexes in axonal cargo vesicles targeted to presynaptic glutamatergic terminals.”

“IQSEC2 (also known as BRAG1/IQ-ArfGEF) is a neuronal ArfGEF for Arf GTPases that typically localizes to excitatory synapses as part of the NMDAR complex, via direct interaction with disks large MAGUK scaffold protein 4 (DLG4/PSD-95/SAP90; Murphy et al. 2006; Sakagami et al. 2008).”

“These auxiliary proteins include the lipoma HMGIC fusion partner-like 3 and 4 (LHFPL3/LHFPL4; Davenport et al. 2017; Yamasaki et al. 2017 Han et al. 2019) or (iv) promoting cell-surface expression while also slowing GABA A R deactivation (TMEM132B; Wang et al. 2024).”

“A GlyR α3L-specific antibody revealed that GlyR α3L clusters at glutamatergic nerve endings and co-localized with VGluT (Eichler et al. 2009).”

“They are integral components of synapse-organizing protein complexes (Kamimura and Maeda 2021) and serve as ligands for leucine-rich repeat transmembrane neuronal proteins (LRRTMs) and leukocyte common antigen-related (LAR) family receptor protein tyrosine phosphatases (RPTPs).”

“IQSEC2 (also known as BRAG1/IQ-ArfGEF) is a neuronal ArfGEF for Arf GTPases that typically localizes to excitatory synapses as part of the NMDAR complex, via direct interaction with disks large MAGUK scaffold protein 4 (DLG4/PSD-95/SAP90; Murphy et al. 2006; Sakagami et al. 2008).”

“…ICD residues 316 RFRRK 320 and 385 KK 386 are known to be critical for binding cytosolic G-protein βγ subunits, which in turn enhance currents mediated by recombinant GlyR α1 and functional allosteric modulation by ethanol at behaviorally relevant concentrations (Yevenes et al. 2006, 2008, 2010).”

“By contrast, IQSEC3 (also known as BRAG3/SynArfGEF) colocalizes with GlyRs and GABA A Rs in the retina and brain (Sakagami et al. 2013), interacts with the gephyrin G domain (van der Spek et al. 2020), utrophin/dystrophin, and S-SCAM/MAGI-2 (Fukaya et al. 2011).”

“…of α2-containing GABA A Rs to the axon initial segment, and disrupting GABA A R α2-collybistin interactions results in increased susceptibility to seizures, early mortality, deficits in working and recognition memory, hyperactivity, anxiety, and reduced social preference (Hines et al. 2018, 2022).”

“…neurobeachin (Lutzenkirchen et al. 2024), neuroligins 2 and 4 (Poulopoulos et al. 2009; Hoon et al. 2011) and importantly the TM3-TM4 ICDs of GABA A R α1, α2, α3 and α5 subunits (Tretter et al. 2008, 2011; Saiepour et al. 2010; Mukherjee et al. 2011; Maric et al. 2011, 2014; Brady and Jacob 2015).”

“…the use of knockout tissue as a negative control; or (iii) Using gene-editing technologies to introduce epitopes (e.g., myc, pHluorin or Venus tags) into the gene/protein of interest (Schwenk et al. 2009, 2012; Heller et al. 2012; Chen et al. 2014; Nakamura et al. 2016; van der Spek et al. 2022).”

“However, recent studies have suggested that native heteromeric GlyRs have an invariant 4α:1β configuration (Yu et al. 2021; Zhu and Gouaux 2021).”

“…the use of knockout tissue as a negative control; or (iii) Using gene-editing technologies to introduce epitopes (e.g., myc, pHluorin or Venus tags) into the gene/protein of interest (Schwenk et al. 2009, 2012; Heller et al. 2012; Chen et al. 2014; Nakamura et al. 2016; van der Spek et al. 2022).”

“…ICD residues 316 RFRRK 320 and 385 KK 386 are known to be critical for binding cytosolic G-protein βγ subunits, which in turn enhance currents mediated by recombinant GlyR α1 and functional allosteric modulation by ethanol at behaviorally relevant concentrations (Yevenes et al. 2006, 2008, 2010).”

“For example, different inhibitory GABA A R subtypes interact with a variety of trans-membrane auxiliary subunits that either regulate receptor trafficking or modulate functional and pharmacological properties (Han et al. 2021).”

“Undeterred, Del Pino et al. (2014) went on to discover a third GlyR β-interacting protein: syndapin I. Syndapin I is a synaptically enriched protein implicated in membrane remodeling, endosomal/ vesicle trafficking, and actin cytoskeletal dynamics (Quan and Robinson 2013).”

“…ICD residues 316 RFRRK 320 and 385 KK 386 are known to be critical for binding cytosolic G-protein βγ subunits, which in turn enhance currents mediated by recombinant GlyR α1 and functional allosteric modulation by ethanol at behaviorally relevant concentrations (Yevenes et al. 2006, 2008, 2010).”

“…the use of knockout tissue as a negative control; or (iii) Using gene-editing technologies to introduce epitopes (e.g., myc, pHluorin or Venus tags) into the gene/protein of interest (Schwenk et al. 2009, 2012; Heller et al. 2012; Chen et al. 2014; Nakamura et al. 2016; van der Spek et al. 2022).”

“As a result, microcephaly was observed in newborn Glra2 knockout mice (Avila et al. 2014), as well as increased susceptibility to seizures (Morelli et al. 2017) and learning and memory defects (Pilorge et al. 2016; Molchanova et al. 2017).”

“Second, the charged residues 316 RFRRKRR 322 are located within a proposed MX-helix (GlyR α1S H311-G329) at the start of the GlyR α1 ICD that governs membrane topology and influences receptor function (Sadtler et al. 2003).”

“IQSEC2 is involved in dendritic spine morphogenesis (Hinze et al. 2017) and knockout mice have revealed defects in AMPAR-, NMDAR-, and GABA A R-mediated synaptic transmission (Mehta et al. 2021) suggesting that IQSEC2 is also important for the function of inhibitory synapses.”

“As a result, microcephaly was observed in newborn Glra2 knockout mice (Avila et al. 2014), as well as increased susceptibility to seizures (Morelli et al. 2017) and learning and memory defects (Pilorge et al. 2016; Molchanova et al. 2017).”

“…expressed in the spinal cord and brainstem, and consistent with this, several spontaneous mouse mutants in the corresponding genes ( Glra1 or Glrb ) exhibit neuromotor defects and develop an exaggerated startle response to acoustic or tactile stimuli at around 2 weeks of age (Schaefer et al. 2013).”

“The isolated collybistin PH domain binds to phosphatidylinositol 3-phosphate (PI3P) that is associated with early/sorting endosomes (Kalscheuer et al. 2009; Papadopoulos et al. 2017).”

“Many of these approaches have focused on using GlyR TM3-TM4 ICDs as baits in glutathione S-transferase (GST) pulldown assays (e.g., Del Pino et al. 2011, 2014; Bluem et al. 2007).”

“With the exception of the importins (Melzer et al. 2010) few studies have investigated interactors of the GlyR α3 subunit, which is surprising given the key roles of this subtype in central inflammatory pain sensitization and rhythmic breathing.”

“Hence, these positively charged residues may electro-statically attract incoming anions to the intracellular compartment (Gibbs et al. 2023).”

“…sites for ubiquitination (Büttner et al. 2001), phosphorylation by protein kinases (e.g., PKC, PKA, tyrosine kinases; Harvey, Depner et al. 2004; Specht et al. 2011; Bai et al. 2024), and importantly for GlyR proteomics, polyproline helix type II (PPII) motifs (Langlhofer and Villmann 2016).”

“Further studies have demonstrated that IQSEC3 is vital for inhibitory synapse density, synaptic transmission, and correct matching of PSDs to pre-synaptic terminals (Um et al. 2016; Früh et al. 2018; Kim et al. 2022).”

“In cell lines, GlyR α3S exhibits a diffuse membrane localization and fast desensitization kinetics, while α3L exhibits a clustered membrane appearance and slow desensitization kinetics (Nikolic et al. 1998; Notelaers et al. 2012, 2014).”

“This system recapitulates the properties of native GlyRs, since recombinant α1β and α3β GlyRs mediate fast decaying inhibitory postsynaptic currents (IPSCs) whereas α2β and α4β GlyRs mediate slow decaying IPSCs (Zhang et al. 2015; Leacock et al. 2018).”

“As a result, microcephaly was observed in newborn Glra2 knockout mice (Avila et al. 2014), as well as increased susceptibility to seizures (Morelli et al. 2017) and learning and memory defects (Pilorge et al. 2016; Molchanova et al. 2017).”

“…in a range of neuro-developmental disorders (NDDs) featuring autism spectrum disorder (ASD), developmental delay (DD), intellectual disability (ID) often accompanied by microcephaly, language delay, and epilepsy (Pilorge et al. 2016; Zhang et al. 2017; Chen et al. 2022; Marcogliese et al. 2022).”

“By contrast, IQSEC3 (also known as BRAG3/SynArfGEF) colocalizes with GlyRs and GABA A Rs in the retina and brain (Sakagami et al. 2013), interacts with the gephyrin G domain (van der Spek et al. 2020), utrophin/dystrophin, and S-SCAM/MAGI-2 (Fukaya et al. 2011).”

“In this regard, it is vital that information on synaptic PPIs is integrated into publicly available platforms such as SynGO (Synaptic Gene Ontology), an expert-curated universal reference for synapse research and an online analysis platform for interpretation of large-scale proteomics data (Koopmans et al. 2019; https…”

“Many of these approaches have focused on using GlyR TM3-TM4 ICDs as baits in glutathione S-transferase (GST) pulldown assays (e.g., Del Pino et al. 2011, 2014; Bluem et al. 2007).”

“IQSEC2 is involved in dendritic spine morphogenesis (Hinze et al. 2017) and knockout mice have revealed defects in AMPAR-, NMDAR-, and GABA A R-mediated synaptic transmission (Mehta et al. 2021) suggesting that IQSEC2 is also important for the function of inhibitory synapses.”

“…15mer peptides with an offset of one amino acid was prepared to map the benchmark interaction of the gephyrin E domain to GlyR β and GABA A R α3 and successfully identified critical motifs in previously known binding sites 395 FNIVG 399 (GABA A R α3) and 420 FSIVG 424 (GlyR β; Schulte et al. 2021).”

“The RhoGEF collybistin localizes to inhibitory synapses and contains an N-terminal SRC homology 3 (SH3) domain, a catalytic RhoGEF domain, and a pleckstrin homology (PH) domain (Kins et al. 2000; Harvey, Duguid et al. 2004; Patrizi et al. 2012).”

“Subunit Interactors—Collybistin, GPR39, HUWE1, Importins α3/α4 and Gβγ”

“Consistent with these findings, missense mutations and deletions affecting the GlyR α2 subunit have been implicated in a range of neuro-developmental disorders (NDDs) featuring autism spectrum disorder (ASD), developmental delay (DD), intellectual disability (ID) often accompanied by microcephaly, language delay, and…”

“Pioneered by Lynch and colleagues, this system induces the formation of artificial synapses between cultured spinal or cortical neurons and HEK293 cells expressing GlyR subunits of interest plus neuroli-gin 2 (Dixon et al. 2015; Zhang et al. 2015).”

“…the use of knockout tissue as a negative control; or (iii) Using gene-editing technologies to introduce epitopes (e.g., myc, pHluorin or Venus tags) into the gene/protein of interest (Schwenk et al. 2009, 2012; Heller et al. 2012; Chen et al. 2014; Nakamura et al. 2016; van der Spek et al. 2022).”

“…GlyR α1 K385A/K386A or GlyR α2 K389A/R390A that disrupt G-protein βγ interactions (Yevenes et al. 2010) had reduced ethanol sensitivity, faster recovery from a sedative dose of ethanol, and higher ethanol intake in the drinking in the dark protocol (Aguayo et al. 2014; Gallegos et al. 2021).”

“This system recapitulates the properties of native GlyRs, since recombinant α1β and α3β GlyRs mediate fast decaying inhibitory postsynaptic currents (IPSCs) whereas α2β and α4β GlyRs mediate slow decaying IPSCs (Zhang et al. 2015; Leacock et al. 2018).”

“High-throughput synaptic interaction proteomics has bur-geoned since the beginning of the century, largely driven by advancements in the accuracy and sensitivity of mass spectroscopy downstream of the affinity purification of solubilized receptor complexes from native tissue (Li et al. 2010).”

“…this system has been used to investigate GlyR α1 and α2 mutations in startle disease and NDDs (Zhang et al. 2016, 2017; Langlhofer et al. 2020; Chen et al. 2022) this unique system has not yet been used to assess the impacts of GlyR interacting proteins such as gephyrin/collybistin or…”

“GlyR α3 knockout and knock-in mice have revealed roles for this subtype in central inflammatory pain sensitization (Harvey, Depner et al. 2004; Werynska et al. 2021) and rhythmic breathing (Manzke et al. 2010).”

“Subunit Interactors—Collybistin, GPR39, HUWE1, Importins α3/α4 and Gβγ”

“However, additional biochemical experiments did not clearly demonstrate that GlyR α1 P366L interferes with colly-bistin binding in vitro (Breitinger et al. 2021).”

“…inhibitory synapses, and artificial or X-linked intellectual disability/epilepsy mutations affecting the SH3 or PH domains cause mis-localization of geph-yrin and inhibitory receptors (Kins et al. 2000; Harvey, Duguid et al. 2004; Kalscheuer et al. 2009; Reddy-Alla et al. 2010; Chiou et al. 2019).”

“However, no clear association of GlyR α1 was observed with im-portins 3 and 4 in immunocytochemistry of spinal cord neuronal cultures (Melzer et al. 2010) and GlyR-importin interactions have not been observed in GlyR immunoprecipitation studies (van der Spek et al. 2020).”

“…there are cases of startle disease that cannot be explained by mutations in known genes (encoding GlyR α1, β, GlyT2 or Asc-1; Schaefer et al. 2022; Drehmann et al. 2023), as well as the identification of the SH3-domain containing in-teractors affected by GlyR α2 P369T and GlyR α2 P373L variants…”

“GlyR α2 also binds Gβγ via similar motifs (ICD residues 316 RFRRK 320 and 385 KR 390 ) but requires co-expression of GlyR β for effective Gβγ and ethanol modulation (Muñoz et al. 2021).”

“However, additional biochemical experiments did not clearly demonstrate that GlyR α1 P366L interferes with colly-bistin binding in vitro (Breitinger et al. 2021).”

“…(3,4)- bisphosphate [PI(3,4)P 2 ], phosphatidylinositol 4,5-bisphosphate [PIP 2 , PI(4,5)P 2 ] and phosphatidylinositol (3,4,5)- trisphosphate [PIP 3 , PI(3,4,5)P 3 ], thereby enabling synaptic collybistin and gephyrin clustering (Soykan et al. 2014; Schäfer et al. 2020; Kilisch et al. 2020).”

“Collybistin exists as multiple splice variants in vivo (CB1, CB2, CB3), some of which lack the SH3 domain (e.g., CB2 SH3-; Harvey, Duguid et al. 2004; George et al. 2021).”

“…can be relieved by the binding of several neuronal interactors to the collybistin SH3 domain, including neuroligins 2 and 4 (Poulopoulos et al. 2009; Hoon et al. 2011), the GABA A R α2 subunit (Saiepour et al. 2010) or the binding of the small GTPase TC10 to the PH domain (Imam et al. 2022).”

“This approach has been employed with great success in the characterization of AMPAR and GABA A R interactomes (Schwenk et al. 2009, 2012; Heller et al. 2012; Chen et al. 2014; Chen, Koopmans, Paliukhovich, et al. 2023; Chen et al. 2023; Nakamura et al. 2016; van der Spek et al. 2022).”

“Neurexins are clearly involved in the maintenance of glycinergic synapses, since triple neurexin 1/2/3 conditional knockout mice reduced the amplitude and altered the kinetics of glycinergic synaptic transmission in the brainstem (Jiang et al. 2024).”

“…missense mutations in the α 1, α 2, and β subunit intracellular domains linked to startle disease (associated with GlyR α 1 and β subunits) and neurodevelopmental disorders such as autism, intellectual disability, and epilepsy, which are specifically linked to the GlyR α 2 subunit [62].”

“Gephyrin interacts with GlyRs, speci fi cally the β -subunit, and with the GABA A R α 1-3 subunits through GephE, which binds to the intracellular loop located between transmembrane helices 3 and 4 of the respective receptor subunits.”

“Evidence from human and murine studies indeed indicates that dysfunction in glycine receptors is associated with epileptic phenotypes, and several receptor subtypes have been proposed as potential therapeutic targets [286–289].”

“Together, these mechanisms enable complex processes such as motor and sensory functions [11–13], as well as learning and memory formation [13–16].”

“Since neither syndapin I nor collybistin appear to directly interact with GlyR α2 56,57 , and the majority of known GlyR interactors bind to the GlyR α1 or β subunits 13 , this highlights a pressing need for high-fidelity proteomic studies of the GlyRs containing the α2-α4 isoforms.”

“Comprehensive reviews and milestone updates on complex receptor-associated protein networks summarize how these diverse structures may influence transmission dynamics [34,164].”

“The activity-dependent regulations of GlyRs number on the plasma membrane and postsynaptic sites are important ways to modulate inhibitory synaptic strength 17,40 .”