The GABAA/benzodiazepine receptor is a heterotetramer of homologous alpha and beta subunits.

C, Mamalaki; Stephenson, F. Anne; Barnard, Eric A.

doi:10.1002/j.1460-2075.1987.tb04791.x

Cited by 99 publications

(53 citation statements)

References 23 publications

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“…Quantitation of the gradients revealed that both proteins exhibited 9 S sedimentation coefficients, as previously described for functional GABA A receptors composed of ␣␤ or ␣␤␥ subunits (Fig. 6 A,B; Mamalaki et al, 1987Mamalaki et al, , 1989Hadingham et al, 1992;Gorrie et al, 1997;Tretter et al, 1997). In contrast, unassembled ␣ or ␤ subunits have 5 S sedimentation coefficients (Gorrie et al, 1997;Tretter et al, 1997).…”

Section: Reduced Oligomerization Of (9e10) ␣1 (Hy) With the ␤3 Subunitsupporting

confidence: 73%

“…Together, these results demonstrate that Q67 and S68 within the ␣1 subunit are critical in mediating assembly with ␤3 to form 9S complexes, representing functional cell surface receptors (Mamalaki et al, 1987(Mamalaki et al, , 1989Hadingham et al, 1992;Gorrie et al, 1997;Tretter et al, 1997). (9E10) ␣1 (HY) subunits appear to oligomerize less efficiently with (FL AG) ␤3 compared to (9E10) ␣1 and are rapidly degraded as previously described for unassembled wild-type ␣1 subunits (Gorrie et al, 1997).…”

Section: Reduced Oligomerization Of (9e10) ␣1 (Hy) With the ␤3 Subunitsupporting

confidence: 73%

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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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Section: Reduced Oligomerization Of (9e10) ␣1 (Hy) With the ␤3 Subunitsupporting

confidence: 73%

Section: Reduced Oligomerization Of (9e10) ␣1 (Hy) With the ␤3 Subunitsupporting

confidence: 73%

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

et al. 2000

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“…Three potential glycosylation sites occur at identical positions (Asn 8, 80 and 149) in the rat and bovine fl-subunit proteins. Glycosylation at any, or all of these sites (and those in the c~-subunit sequence) may account for the discrepancy between the predicted and observed Mr values of cDNA-derived [7,8] and biochemically isolated [1][2][3][4][5][6] GABAA receptor subunits, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…The GABAA receptor has been purified by affinity chromatography from bovine, porcine, chick and rat brain [1][2][3][4][5]. The predominant GABAA receptor in cerebral cortex is comprised of /5'-and ~-subunit polypeptides, which bind GABA (or GABAA agonists, muscimol and isoguvacine) and benzodiazepines (e.g.…”

Section: Introductionmentioning

confidence: 99%

Cloning and expression of a novel rat GABA_A receptor

Lolait

O’Carroll

Kusano³

et al. 1989

FEBS Letters

105

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Two full-length cDNA clones encoding ~t-and fl-subunits of a GABAA receptor have been isolated from a rat cerebral cortex eDNA library. The mature ~t-subunit protein consists of 428 amino acids with a calculated Mr of 48680. This protein is highly homologous (~ 99% amino acid identity) with the bovine brain ~q-subunit receptor [(1988) Nature 335, 76-79]. The mature rat fl-subunit receptor is a 448 amino acid polypeptide and shares ~ 80% amino acid identity with the previously characterized bovine GABAA receptor fl-subunit [(1987) Nature 328, 221-227]. Co-expression of the cloned DNA in Xenopus oocytes produces a functional receptor and ion channel with pharmacological characteristics of a GABAA receptor. GABAA ~t-and fl-subunit mRNA is detectable in the cortex, cerebellum and hippocampus.

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“…Anti-␤2 subunit antibodies recognized only two bands with M r 56,000 and 58,000 in forebrain but three to four bands in solubilized preparations of transfected HEK 293 cells. Deglycosylation of native GABA A receptor ␤2 subunits yielded two additional immunoreactive bands (39). Thus, because the ␤2 subunit is overexpressed in the HEK 293 cells, it is probable that the two additional bands are non-N-glycosylated forms of the ␤2 subunit.…”

Section: Grif-1 a Novel Gaba A Receptor-associated Proteinmentioning

confidence: 99%

Identification, Molecular Cloning, and Characterization of a Novel GABAA Receptor-associated Protein, GRIF-1

Beck¹,

Brickley²,

Wilkinson³

et al. 2002

Journal of Biological Chemistry

103

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A novel 913-amino acid protein, ␥-aminobutyric acid type A (GABA A ) receptor interacting factor-1 (GRIF-1), has been cloned and identified as a GABA A receptorassociated protein by virtue of its specific interaction with the GABA A receptor ␤2 subunit intracellular loop in a yeast two-hybrid assay. GRIF-1 has no homology with proteins of known function, but it is the rat orthologue of the human ALS2CR3/KIAA0549 gene. GRIF-1 is expressed as two alternative splice forms, GRIF-1a and a C-terminally truncated form, GRIF-1b. GRIF-1 mRNA has a wide distribution with a major transcript size of 6.2 kb. GRIF-1a protein is only expressed in excitable tissues, i.e. brain, heart, and skeletal muscle major immunoreactive bands of M r ϳ 115 and 106 kDa and, in muscle and heart only, an additional 88-kDa species. When expressed in human embryonic kidney 293 cells, GRIF-1a yielded three immunoreactive bands with M r ϳ 115, 106, and 98 kDa. Co-expression of GRIF-1a and ␣1␤2␥2 GABA A receptors in mammalian cells revealed some co-localization in the cell cytoplasm. Anti-FLAGagarose specifically precipitated GRIF-1 FLAG and GABA A receptor ␤2 subunits from human embryonic kidney 293 cells co-transfected with GRIF-1a FLAG and ␤2 subunit clones. Further, immobilized GRIF-1-(8 -633) specifically precipitated in vitro GABA A receptor ␣1 and ␤2 subunit immunoreactivities from detergent extracts of adult rat brain. The respective GABA A receptor ␤2 subunit/GRIF-1 binding domains were mapped using the yeast two-hybrid reporter gene assays. A possible role for GRIF-1 as a GABA A receptor ␤2 subunit trafficking factor is proposed.

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The GABAA/benzodiazepine receptor is a heterotetramer of homologous alpha and beta subunits.

Cited by 99 publications

References 23 publications

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

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

Cloning and expression of a novel rat GABA_A receptor

Identification, Molecular Cloning, and Characterization of a Novel GABAA Receptor-associated Protein, GRIF-1

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The GABAA/benzodiazepine receptor is a heterotetramer of homologous alpha and beta subunits.

Cited by 99 publications

References 23 publications

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

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

Cloning and expression of a novel rat GABAA receptor

Identification, Molecular Cloning, and Characterization of a Novel GABAA Receptor-associated Protein, GRIF-1

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

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

Cloning and expression of a novel rat GABA_A receptor