α4 is a major acetylcholine binding subunit of cholinergic ligand affinity-purified nicotinic acetylcholine receptor from rat brains

Nakayama, Hitoshi; Nakashima, Toshikatsu; Kurogochi, Yutaka

doi:10.1016/0304-3940(91)90664-f

Cited by 26 publications

(12 citation statements)

References 18 publications

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“…Studies have shown that cr4 is one of the most abundant nAChR subunits in the brain, and that receptors with high affinity for agonist are composed of cr4 and 732 subunits (Wada et al, 1989;Nakayama et al, 1991;Flores et al, 1992). In addition, receptors containing cr4 and 732 subunits may mediate the agonist-evoked release of acetylcholine, become inactivated for a prolonged period of time following sustained nicotine exposure, are more sensitive to nicotine-induced inactivation, and exhibit an increased density following chronic exposure to nicotine (Flores et a!., 1992;Wi!kie et a!., 1993;Yu and Wecker, 1994;Hsu et al, 1996).…”

Section: Resultsmentioning

confidence: 99%

Nicotine Enhances the Cyclic AMP‐Dependent Protein Kinase‐Mediated Phosphorylation of α4 Subunits of Neuronal Nicotinic Receptors

Hsu

Edwards

Wecker

1997

Journal of Neurochemistry

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Studies determined whether α4β2 or α3β2 neuronal nicotinic receptors expressed in Xenopus oocytes are substrates for cyclic AMP‐dependent protein kinase (PKA) and whether nicotine affects receptor phosphorylation. The cRNAs for the subunits were coinjected into oocytes, and cells were incubated for 24 h in the absence or presence of nicotine (50 nM for α4β2 and 500 nM for α3β2 receptors). Nicotine did not interfere with the isolation of the receptors. When receptors isolated from oocytes expressing α4β2 receptors were incubated with [γ‐32P]ATP and the catalytic subunit of PKA, separated by electrophoresis, and visualized by autoradiography, a labeled phosphoprotein with the predicted molecular size of the α4 subunit was present. Phosphorylation of α4 subunits of α4β2 receptors increased within the first 5 min of incubation with nicotine and persisted for 24 h. In contrast, receptors isolated from oocytes expressing α3β2 receptors did not exhibit a labeled phosphoprotein corresponding to the size of the α3 subunit. Results suggest that the PKA‐mediated phosphorylation of α4 and not α3 subunits may explain the differential inactivation by nicotine of these receptors subtypes expressed in oocytes.

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Section: Resultsmentioning

confidence: 99%

Nicotine Enhances the Cyclic AMP‐Dependent Protein Kinase‐Mediated Phosphorylation of α4 Subunits of Neuronal Nicotinic Receptors

Hsu

Edwards

Wecker

1997

Journal of Neurochemistry

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“…Studies have shown that α4 is one of the most abundant neuronal nicotinic receptor subunits in the brain, and that receptors with high affinity for agonist are composed of α4 and β2 subunits (Wada et al . 1989; Nakayama et al . 1991; Flores et al .…”

Section: Discussionmentioning

confidence: 99%

Cyclic AMP‐dependent protein kinase (PKA) and protein kinase C phosphorylate sites in the amino acid sequence corresponding to the M3/M4 cytoplasmic domain of α4 neuronal nicotinic receptor subunits

Wecker

Guo

Jr.

et al. 2001

Journal of Neurochemistry

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To determine whether a4 subunits of a4b2 neuronal nicotinic receptors are phosphorylated within the M3/M4 intracellular region by cyclic AMP-dependent protein kinase A (PKA) or protein kinase C (PKC), immunoprecipitated receptors from Xenopus oocytes and a fusion protein corresponding to the M3/M4 cytoplasmic domain of a4 (a4 3362597 ) were incubated with ATP and either PKA or PKC. Both a4 and a4 3362597 were phosphorylated by PKA and PKC, providing the ®rst direct biochemical evidence that the M3/M4 cytoplasmic domain of neuronal nicotinic receptor a4 subunits is phosphorylated by both kinases. When the immunoprecipitated receptors and the a4 3362597 fusion protein were phosphorylated and the labeled proteins subjected to phosphoamino acid analysis, results indicated that a4 and a4 3362597 were phosphorylated on the same amino acid residues by each kinase. Furthermore, PKA phosphorylated serines exclusively, whereas PKC phosphorylated both serines and threonines. To determine whether Ser 368 was a substrate for both kinases, a peptide corresponding to amino acids 356±371 was synthesized (a4 3562371 ) and incubated with ATP and the kinases. The phosphorylation of a4 3562371 by both PKA and PKC was saturable with K m s of 15.3^3.3 mM and 160.8^26.8 mM, respectively, suggesting that Ser 368 was a better substrate for PKA than PKC.

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“…Cerebellar synaptosomes and Lubrol extracts were prepared from frozen rat brains (PelFreez) as described by Nakayama et al (1991). Regional dissections of rat brain were accomplished by cutting the outermost sections of a fresh ice-cold rat brain away from the center.…”

Section: Methodsmentioning

confidence: 99%

“…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).…”

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confidence: 98%

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

Forsayeth

Kobrin

1997

J. Neurosci.

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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...

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α4 is a major acetylcholine binding subunit of cholinergic ligand affinity-purified nicotinic acetylcholine receptor from rat brains

Cited by 26 publications

References 18 publications

Nicotine Enhances the Cyclic AMP‐Dependent Protein Kinase‐Mediated Phosphorylation of α4 Subunits of Neuronal Nicotinic Receptors

Nicotine Enhances the Cyclic AMP‐Dependent Protein Kinase‐Mediated Phosphorylation of α4 Subunits of Neuronal Nicotinic Receptors

Cyclic AMP‐dependent protein kinase (PKA) and protein kinase C phosphorylate sites in the amino acid sequence corresponding to the M3/M4 cytoplasmic domain of α4 neuronal nicotinic receptor subunits

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

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