Subcellular Localization and Endocytosis of Homomeric γ2 Subunit Splice Variants of γ-Aminobutyric Acid Type A Receptors

Connolly, Christopher N.; Uren, Julia M.; Thomas, P. C.; Gorrie, George; Gibson, Adele; Smart, Trevor G.; Moss, Stephen J.

doi:10.1006/mcne.1999.0746

Cited by 74 publications

(75 citation statements)

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“…This is in contrast to the finding that receptors composed of either ␣ 1 ␤ 2 or ␣ 1 ␤ 2 ␥ 2 L subunits are expressed on the cell surface, but that only those containing a ␥ subunit undergo endocytosis (4,10). Although our results indicate that receptor endocytosis can proceed in the absence of a ␥ subunit, it is possible that the ␥ subunit may play a modulatory role in controlling the rate of endocytosis or receptor trafficking subsequent to endocytosis.…”

Section: Discussioncontrasting

confidence: 99%

“…Antibody-promoted internalization of other integral membrane proteins has been observed under certain conditions (28). Although this possibility cannot be definitively ruled out, we consider it unlikely because the binding of anti-myc antibodies to cell surface GABA A receptors in living HEK 293 cells does not appear to induce GABA A receptor endocytosis (4,8,10). Alternatively, hormones or other signaling molecules present in the serum could affect signal transduction pathways that regulate endocytic processes.…”

Section: Discussionmentioning

confidence: 97%

“…For these experiments, colocalized Texas Red and Alexa 488 labeling represents those Texas Red-labeled receptors that remained on the cell surface during the 37°C incubation, whereas Texas Red labeling not colocalized with Alexa 488 represents receptors that were endocytosed during the 37°C incubation period. The immunolabeling of myc-tagged GABA A receptors in living HEK 293 cells has been used previously to study GABA A receptor endocytosis (4,8,10).…”

Section: Immunofluorescencementioning

confidence: 99%

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Constitutive GABAA Receptor Endocytosis Is Dynamin-mediated and Dependent on a Dileucine AP2 Adaptin-binding Motif within the β2 Subunit of the Receptor

Herring

Huang

Singh

et al. 2003

Journal of Biological Chemistry

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Receptor endocytosis is an important mechanism for regulating the synaptic efficacy of neurotransmitters. There is strong evidence that GABA A receptor endocytosis is clathrin-dependent; however, this process is not well understood. Here we demonstrate that in HEK 293 cells, endocytosis of GABA A receptors composed of either ␣ 1 ␤ 2 ␥ 2 L or ␣ 1 ␤ 2 subunits is blocked by the dominant negative dynamin construct K44A. Furthermore, we identify a dileucine AP2 adaptin-binding motif within the receptor ␤ 2 subunit that is critical for endocytosis. Internalization of GABA A receptors lacking this motif is dramatically inhibited, and the receptors appear to accumulate on the cell surface. Patch clamp analysis of receptors lacking the dileucine motif show that there is an increase in the peak amplitude of GABA-gated chloride currents compared with wild-type receptors. Additionally, GABA-gated chloride currents in HEK 293 cells expressing wild-type receptors are increased by introduction of a peptide corresponding to the dileucine motif region of the receptor ␤ 2 subunit but not by a control peptide containing alanine substitutions for the dileucine motif. In mouse brain cerebral cortical neurons, the dileucine motif peptide increases GABA-gated chloride currents of native GABA A receptors. This is the first report to our knowledge that an AP2 adaptin dileucine recognition motif is critical for the endocytosis of ligand-gated ion channels belonging to this superfamily.The GABA A receptor is a ligand-gated chloride channel that, upon activation by GABA 1 (␥-aminobutyric acid), mediates increases in chloride conductance resulting in membrane hyperpolarization and neuronal inhibition (1). The role of these receptors in hyperexcitability states, such as epilepsy and anxiety, is widely recognized. Importantly, GABA A receptors mediate the effects of benzodiazepines and barbiturates, two frequently prescribed classes of therapeutic agents. The GABA A receptor is a pentameric receptor composed of multiple subunits, each containing four membrane-spanning regions (M1-M4) with a large intracellular loop between M3 and M4. A number of subunits exist (␣ 1Ϫ6 , ␤ 1Ϫ3 , ␥ 1Ϫ3 , ␦, , ⑀, ), and receptors composed of ␣ 1 ␤ 2 ␥ 2 L subunits are believed to represent the predominant GABA A receptor subtype in the brain (1).Receptor endocytosis is known to regulate the cell surface expression of neurotransmitter receptors, and such regulation is an important mechanism for controlling the synaptic efficacy of neurotransmitters (2). Although GABA A receptors undergo endocytosis, the mechanism is not well understood. Several lines of evidence indicate that GABA A receptor endocytosis may be clathrin/dynamin-dependent. These include the presence of GABA A receptors in clathrin-coated vesicles isolated from brain (3), the colocalization of the receptor with transferrin receptors (4), and the colocalization and co-immunoprecipitation of hippocampal GABA A receptors with the clathrin adaptor complex AP2 adaptin (5). Additionally, peptides that dis...

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Section: Discussioncontrasting

confidence: 99%

Section: Discussionmentioning

confidence: 97%

Section: Immunofluorescencementioning

confidence: 99%

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Constitutive GABAA Receptor Endocytosis Is Dynamin-mediated and Dependent on a Dileucine AP2 Adaptin-binding Motif within the β2 Subunit of the Receptor

Herring

Huang

Singh

et al. 2003

Journal of Biological Chemistry

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“…3B; Connolly et al, 1996). Similar migration of the ␥2 subunit has been seen previously in recombinant preparations and for the ␥2 subunit expressed in neuronal membranes (Stephenson et al, 1990;Connolly et al, 1996Connolly et al, , 1999Tretter et al, 1997). Both (9E10) ␣1 and (9E10) ␣1S coprecipitated with (FL AG) ␥2L at similar levels (Fig.…”

Section: Amino Acids 58-67 Control the Oligomerization Of Gaba A Recesupporting

confidence: 83%

Identification of Residues within GABA_AReceptor α Subunits That Mediate Specific Assembly with Receptor β Subunits

et al. 2000

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GABA A receptors can be constructed from a range of differing subunit isoforms: ␣, ␤, ␥, ␦, and ⑀. Expression studies have revealed that production of GABA-gated channels is achieved after coexpression of ␣ and ␤ subunits. The expression of a ␥ subunit isoform is essential to confer benzodiazepine sensitivity on the expressed receptor. However, how the specificity of subunit interactions is controlled during receptor assembly remains unknown. Here we demonstrate that residues 58-67 within ␣ subunit isoforms are important in the assembly of receptors comprised of ␣␤ and ␣␤␥ subunits. Deletion of these residues from the ␣1 or ␣6 subunits results in retention of either ␣ subunit isoform in the endoplasmic reticulum on coexpression with the ␤3, or ␤3 and ␥2 subunits. Immunoprecipitation revealed that residues 58-67 mediated oligomerization of the ␣1 and ␤3 subunits, but were without affect on the production of ␣/␥ complexes. Within this domain, glutamine 67 was of central importance in mediating the production of functional ␣1␤3 receptors. Mutation of this residue resulted in a drastic decrease in the cell surface expression of ␣1␤3 receptors and the resulting expression of ␤3 homomers. Sucrose density gradient centrifugation revealed that this residue was important for the production of a 9S ␣1␤3 complex representing functional GABA A receptors.Therefore, our studies detail residues that specify GABA A receptor ␣␤ subunit interactions. This domain, which is conserved in all ␣ subunit isoforms, will therefore play a critical role in the assembly of GABA A receptors composed of ␣␤ and ␣␤␥ subunits. Key words: GABA A -receptor; assembly; cell surface expression; N-terminal; oligomerization; ␣ subunitGABA A receptors are critical mediators of fast synaptic inhibition in the brain and are also important drug targets for a range of compounds, including the benzodiazepines and barbiturates (MacDonald and Olsen, 1994;Rabow et al., 1995). GABA A receptors are members of the ligand-gated ion channel superfamily that includes glycine, nicotinic acetylcholine (AChR), and 5-HT 3 receptors (Unwin, 1993). Molecular cloning has revealed a range of GABA A receptor subunits that can be divided by homology into subunit classes with multiple members: ␣(1-6), ␤ (1-3), ␥(1-3), ␦, ⑀, and (MacDonald and Olsen, 1994;Rabow et al., 1995;Davies et al., 1997;Hedblom and Kirkness, 1997). There is considerable spatial and temporal variation in subunit expression, with many neuron types expressing multiple numbers of receptor subunits (Laurie et al., 1992;MacDonald and Olsen, 1994;Rabow et al., 1995). Clearly, to delineate the true diversity of GABA A receptor structure in the brain, it is important to gain some insights into how these receptor subunits are assembled.Studies using heterologous expression focusing on the receptor ␣1, ␤1-2, and ␥2 subunits, have revealed that access to the cell surface is limited to the combinations ␣␤ and ␣␤␥2 (Angelotti and MacDonald 1993;MacDonald and Olsen, 1994;Rabow et al., 1995;Connolly et al., 1996). Most s...

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“…We therefore quantified the concentrations of different types of antidepressants and antipsychotics in cell fractions of human embryonic kidney 293 (HEK 293) cells stably transfected with the 5-HT3 A receptor and of N1E-115 neuroblastoma cells in relation to the membraneous localization of the 5-HT 3 receptor protein. Because receptor internalization is a major principle of the downregulation of the GABA A receptor (Tehrani and Barnes, 1997;Connolly et al, 1999) that shares structural features with the 5-HT 3 receptor, we investigated whether the antagonistic effects of antidepressants at the 5-HT 3 receptor are also conferred via an enhancement of receptor internalization.…”

Section: Introductionmentioning

confidence: 99%

Antidepressants and Antipsychotic Drugs Colocalize with 5-HT₃Receptors in Raft-Like Domains

Eisensamer¹,

Uhr²,

Meyr³

et al. 2005

J. Neurosci.

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Subcellular Localization and Endocytosis of Homomeric γ2 Subunit Splice Variants of γ-Aminobutyric Acid Type A Receptors

Cited by 74 publications

References 0 publications

Constitutive GABAA Receptor Endocytosis Is Dynamin-mediated and Dependent on a Dileucine AP2 Adaptin-binding Motif within the β2 Subunit of the Receptor

Constitutive GABAA Receptor Endocytosis Is Dynamin-mediated and Dependent on a Dileucine AP2 Adaptin-binding Motif within the β2 Subunit of the Receptor

Identification of Residues within GABA_AReceptor α Subunits That Mediate Specific Assembly with Receptor β Subunits

Antidepressants and Antipsychotic Drugs Colocalize with 5-HT₃Receptors in Raft-Like Domains

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Subcellular Localization and Endocytosis of Homomeric γ2 Subunit Splice Variants of γ-Aminobutyric Acid Type A Receptors

Cited by 74 publications

References 0 publications

Constitutive GABAA Receptor Endocytosis Is Dynamin-mediated and Dependent on a Dileucine AP2 Adaptin-binding Motif within the β2 Subunit of the Receptor

Constitutive GABAA Receptor Endocytosis Is Dynamin-mediated and Dependent on a Dileucine AP2 Adaptin-binding Motif within the β2 Subunit of the Receptor

Identification of Residues within GABAAReceptor α Subunits That Mediate Specific Assembly with Receptor β Subunits

Antidepressants and Antipsychotic Drugs Colocalize with 5-HT3Receptors in Raft-Like Domains

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Identification of Residues within GABA_AReceptor α Subunits That Mediate Specific Assembly with Receptor β Subunits

Antidepressants and Antipsychotic Drugs Colocalize with 5-HT₃Receptors in Raft-Like Domains