Properties of embryonic and adult muscle acetylcholine receptors transiently expressed in COS cells

Gu, Yun; Franco, Alfredo; Gardner, Paul; Lansman, Jeffry B.; Forsayeth, John; Hall, Zach W.

doi:10.1016/0896-6273(90)90305-y

Cited by 90 publications

(79 citation statements)

References 51 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2). The apparent molecular mass and breadth of the band were consistent with those previously seen for the ␦ subunit expressed either normally in C2 muscle cells (15) or following transfection in COS cells (31). Because the glycosylation-incompetent G1G2G3 mutant also ran as a doublet (Figs.…”

Section: Methodssupporting

confidence: 67%

“…All of the ␦ proteins appeared on SDS gels either as a broad diffuse band or as a doublet, as previously reported for the ␦ subunit in AChRs of adult muscle cells (20), cultured myotubes (37,38), or transfected heterologous cells (31). The breadth and/or multiplicity of the bands has been thought to be due to variable glycosylation (21,38), but this explanation cannot account for the fact that the unglycosylated peptide also appears to be heterogenous.…”

Section: Discussionmentioning

confidence: 65%

See 1 more Smart Citation

Altered Glycosylation Sites of the δ Subunit of the Acetylcholine Receptor (AChR) Reduce αδ Association and Receptor Assembly

Ramanathan

Hall²

1999

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

We have used mutagenesis to investigate the potential N-glycosylation sites in the ␦ subunit of the mouse muscle acetylcholine receptor (AChR). Of the three sites, Asn 76 , Asn 143 , and Asn 169 , only the first two were glycosylated when the ␦ subunit was expressed in COS cells. Because the heterologously expressed ␦ subunit was similar in its properties to that expressed in C2 muscle cells, the sites of glycosylation are likely to be the same in both cases. In COS cells, mutations of the ␦ subunit that prevented glycosylation at either of the sites did not change its metabolic stability nor its steady-state level. These results are in contrast to those found previously for the ␣ subunit, in which glycosylation at a single site metabolically stabilized the polypeptide (Blount, P., and Merlie, J. P. (1990) J. Cell Biol. 111, 2613-2622). Mutations of the ␦ subunit that prevented glycosylation, however, decreased its ability to form an ␣␦ heterodimer when the ␣ and ␦ subunit were expressed together. When all four subunits of the AChR (␣, ␤, ␦, and ⑀) were coexpressed, mutation of the ␦ subunit to prevent glycosylation resulted in a reduced amount of fully assembled AChR and reduced surface AChR levels, consistent with the role of the heterodimer in the assembly reaction. These results suggest that glycosylation of the ␦ subunit at both Asn 76 and Asn 143 is needed for its efficient folding and/or its subsequent interaction with the ␣ subunit.The muscle nicotinic acetylcholine receptor (AChR), 1 a heterooligomeric membrane receptor, is the best understood member of a family of closely related ligand-gated ion channels that include the neuronal acetylcholine, GABA A , glycine, and serotonin 5-HT 3 receptors (1, 2). These receptors are clustered in the postsynaptic membrane, where they mediate rapid excitatory and inhibitory synaptic transmission. For each member of this family, the individual subunits are arranged in a pseudosymmetric array around an aqueous channel. Binding of the ligand opens the channel, allowing ions to flow through, thus changing the potential of the membrane (3, 4).The adult muscle nicotinic AChR is a pentamer of four subunits, ␣, ␤, ␦, and ⑀, with the stoichiometry ␣ 2 ␤␦⑀ (reviewed in Refs. 3-5). The subunits, which are homologous, each comprise four transmembrane domains with a large extracellular Nterminal domain, a large intracellular loop between the third and fourth transmembrane domains, and a small extracellular C-terminal domain (Fig. 1A) (6 -9). Each of the subunits is synthesized from a separate mRNA, and each undergoes translocation into the endoplasmic reticulum, where it undergoes post-translational processing, including signal sequence cleavage and glycosylation (10). Following synthesis, the ␣ subunit undergoes a folding reaction that appears to involve disulfide bond formation and that confers upon the ␣ subunit the ability to bind ␣-bungarotoxin (␣-BuTx; Refs. 11-13). The other subunits presumably undergo folding reactions as well, although it has not been possible to measur...

show abstract

Section: Methodssupporting

confidence: 67%

Section: Discussionmentioning

confidence: 65%

Altered Glycosylation Sites of the δ Subunit of the Acetylcholine Receptor (AChR) Reduce αδ Association and Receptor Assembly

Ramanathan

Hall²

1999

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Reconstitution of nicotinic receptors in transiently transfected COS cells has been used considerably to study assembly of the muscle nicotinic receptor (Gu et al, 1990(Gu et al, , 1991Forsayeth et al, 1992). Thus, COS cells were transfected with various combinations of subunits, and acetylcholine-binding activity was measured in the cells.…”

Section: Reconstitution Of Nicotinic Receptors In Cos Cellsmentioning

confidence: 99%

Formation of Oligomers Containing the β3 and β4 Subunits of the Rat Nicotinic Receptor

Forsayeth

Kobrin

1997

J. Neurosci.

Self Cite

View full text Add to dashboard Cite

The role of the ␤3 and ␤4 subunits of the nicotinic acetylcholine receptor in brain is still unclear. We investigated nicotinic receptor structure with antibodies directed against unique regions of the ␤3 and ␤4 subunits of the rat nicotinic acetylcholine receptor. Anti-␤4 detected a single band of 66 kDa in most regions of the brain that was strongest in striatum and cerebellum. The 60 kDa ␤3 subunit was detected primarily in striatum and cerebellum, and faintly in hippocampus. Immunoprecipitation experiments established that the two subunits were coassembled in the cerebellum along with the ␤2 subunit. Antibodies against the ␣4, ␤2, ␤3, and ␤4 subunits immunoprecipitated ϳ75% of the bungarotoxin-insensitive nicotinic receptor from cerebellar extracts as determined by nicotine-dependent acetylcholine binding. Transfection of COS cells with cDNAs for these four subunits induced expression of a high affinity nicotinic receptor. Omission of only a single subunit from the transfection affected either the B max or the apparent K D of the receptor. Our data suggest that the ␤3 subunit functions as a structural entity that links a relatively unstable ␣4␤2 heterodimer to a more stable ␣4␤4 heterodimer. The agonistbinding site formed by ␣4␤2 has a much greater affinity than does that formed by ␣4␤4. In this respect, nicotinic receptors that contain the ␤3 subunit are structurally homologous to the muscle nicotinic receptor.Key words: nicotine; acetylcholine; receptor; antibody; ␤3; ␤4; subunit Nicotinic acetylcholine receptors (nAChR) expressed in the CNS are members of a superfamily of ligand-gated ion channels that also include the muscle-type nAChR, GABA A , glycine, and 5-HT 3 receptors (Barnard et al., 1987;Egebjerg et al., 1991;Maricq et al., 1991;Moriyoshi et al., 1991;Monyer et al., 1992). Nine different rat neuronal nAChR subunit cDNAs (␣2, ␣3, ␣4, ␣5, ␣6, ␣7, ␤2, ␤3, and ␤4) have been cloned (for review, see Sargent, 1993). Most of the receptors in the family are thought to contain two or more different subunits, but the precise combinations of these subunits that exist in vivo are largely unknown. Previous work has indicated that the major high affinity nicotinic receptor in rat brain is composed of ␣4 and ␤2 subunits (Nakayama et al., 1991;Flores et al., 1992;Marks et al., 1992). More recent evidence indicates that deletion of the ␤2 subunit gene results in the loss of high affinity nicotine-binding sites from mouse brain (Picciotto et al., 1995). Nicotinic receptors have also been reconstituted in Xenopus oocytes. Thus, the ␤2 and ␤4 subunits can combine with various ␣ subunits to form functional receptors, permitting the formation of many types of receptor with unique pharmacological characteristics (Luetje and Patrick, 1991). Much less is known about the ␤3 subunit. It does not express any channel activity in oocytes in combination with any other single subunit , nor has it been demonstrated at the protein level in the CNS. Both the ␤3 and ␤4 subunits seem, by in situ hybridization, to have a more restric...

show abstract

“…Alternatively, this short half-life could be the result of a defective interaction between the α7 AChRs proteins needed for long-term receptor stability. Gu et al [36] suggested that the metabolic stability of the AChR arises from its association with other proteins that stabilize it, which are presumably absent from SHE-P1 cells. In our experiment, the dissociation constant K D for αBTX binding to the α7 AChR was rather high compared to previously published values [37,38] .…”

Section: Discussionmentioning

confidence: 99%

Ric-3 chaperone-mediated stable cell-surface expression of the neuronal α7 nicotinic acetylcholine receptor in mammalian cells

2009

View full text Add to dashboard Cite

Aim: Studies of the α7-type neuronal nicotinic acetylcholine receptor (AChR), one of the receptor forms involved in many physiologically relevant processes in the central nervous system, have been hampered by the inability of this homomeric protein to assemble in most heterologous expression systems. In a recent study, it was shown that the chaperone Ric-3 is necessary for the maturation and functional expression of α7-type AChRs [1] . The current work aims at obtaining and characterizing a cell line with high functional expression of the human α7 AChR. Methods: Ric-3 cDNA was incorporated into SHE-P1-hα7 cells expressing the α7-type AChR. Functional studies were undertaken using single-channel patch-clamp recordings. Equilibrium and kinetic [ 125 I]α-bungarotoxin binding assays, as well as fluorescence microscopy using fluorescent α-bungarotoxin, anti-α7 antibody, and GFP-α7 were performed on the new clone. Results: The human α7-type AChR was stably expressed in a new cell line, which we coined SHE-P1-hα7-Ric-3, by co-expression of the chaperone Ric-3. Cell-surface AChRs exhibited [ 125 I]αBTX saturable binding with an apparent K D of about 55 nmol/L. Fluorescence microscopy revealed dispersed and micro-clustered AChR aggregates at the surface of SHE-P1-hα7-Ric-3 cells. Larger micron-sized clusters were observed in the absence of receptor-clustering proteins or upon aggregation with anti-α7 antibodies. In contrast, chaperone-less SHE-P1-hα7 cells expressed only intracellular α7 AChRs and failed to produce detectable single-channel currents. Conclusion:The production of a stable and functional cell line of neuroepithelial lineage with robust cell-surface expression of neuronal α7-type AChR, as reported here, constitutes an important advance in the study of homomeric receptors in mammalian cells.

show abstract

Properties of embryonic and adult muscle acetylcholine receptors transiently expressed in COS cells

Cited by 90 publications

References 51 publications

Altered Glycosylation Sites of the δ Subunit of the Acetylcholine Receptor (AChR) Reduce αδ Association and Receptor Assembly

Altered Glycosylation Sites of the δ Subunit of the Acetylcholine Receptor (AChR) Reduce αδ Association and Receptor Assembly

Formation of Oligomers Containing the β3 and β4 Subunits of the Rat Nicotinic Receptor

Ric-3 chaperone-mediated stable cell-surface expression of the neuronal α7 nicotinic acetylcholine receptor in mammalian cells

Contact Info

Product

Resources

About