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Tyrosine phosphorylation of nicotinic acetylcholine receptor mediates Grb2 binding.

ABSTRACT: Tyrosine phosphorylation of the nicotinic acetylcholine receptor (AChR) is associated with an altered rate of receptor desensitization and also may play a role in agrin-induced receptor clustering. We have demonstrated a previously unsuspected interaction between Torpedo AChR and the adaptor protein Grb2. The binding is mediated by the Src homology 2 (SH2) domain of Grb2 and the tyrosine-phosphorylated delta subunit of the AChR. Dephosphorylation of the delta subunit abolishes Grb2 binding. A cytoplasmic domain of the delta subunit contains a binding motif (pYXNX) for the SH2 domain of Grb2. Indeed, a phosphopeptide corresponding to this region of the delta subunit binds to Grb2 SH2 fusion proteins with relatively high affinity, whereas a peptide lacking phosphorylation on tyrosine exhibits no binding. Grb2 is colocalized with the AChR on the innervated face of Torpedo electrocytes. Furthermore, Grb2 specifically copurifies with AChR solubilized from postsynaptic membranes. These data suggest a novel role for tyrosine phosphorylation of the AChR in the initiation of a Grb2-mediated signaling cascade at the postsynaptic membrane.

SUBMITTER: Colledge M

PROVIDER: S-EPMC6573310 | biostudies-literature | 1997 Jul

REPOSITORIES: biostudies-literature

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Tyrosine phosphorylation of nicotinic acetylcholine receptor mediates Grb2 binding.

Colledge M M Froehner S C SC

The Journal of neuroscience : the official journal of the Society for Neuroscience 19970701 13

Tyrosine phosphorylation of the nicotinic acetylcholine receptor (AChR) is associated with an altered rate of receptor desensitization and also may play a role in agrin-induced receptor clustering. We have demonstrated a previously unsuspected interaction between Torpedo AChR and the adaptor protein Grb2. The binding is mediated by the Src homology 2 (SH2) domain of Grb2 and the tyrosine-phosphorylated delta subunit of the AChR. Dephosphorylation of the delta subunit abolishes Grb2 binding. A cy ...[more]

PMID: 9185541

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Project description:Nicotine, a component of tobacco, is highly addictive but possesses beneficial properties such as cognitive improvements and memory maintenance. Involved in these processes is the neuronal nicotinic acetylcholine receptor (nAChR) alpha7, whose activation triggers depolarization, intracellular signaling cascades, and synaptic plasticity underlying addiction and cognition. It is therefore important to investigate intracellular mechanisms by which a cell regulates alpha7 nAChR activity. We have examined the role of phosphorylation by combining molecular biology, biochemistry, and electrophysiology in SH-SY5Y neuroblastoma cells, Xenopus oocytes, rat hippocampal interneurons, and neurons from the supraoptic nucleus, and we found tyrosine phosphorylation of alpha7 nAChRs. Tyrosine kinase inhibition by genistein decreased alpha7 nAChR phosphorylation but strongly increased acetylcholine-evoked currents, whereas tyrosine phosphatase inhibition by pervanadate produced opposite effects. Src-family kinases (SFKs) directly interacted with the cytoplasmic loop of alpha7 nAChRs and phosphorylated the receptors at the plasma membrane. SFK inhibition by PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] or SU6656 (2,3-dihydro-N,N-dimethyl-2-oxo-3-[(4,5,6,7-tetrahydro-1H-indol-2-yl)methylene]-1H-indole-5-sulfonamide) increased alpha7 nAChR-mediated responses, whereas expression of active Src reduced alpha7 nAChR activity. Mutant alpha7 nAChRs lacking cytoplasmic loop tyrosine residues because of alanine replacement of Tyr-386 and Tyr-442 were more active than wild-type receptors and insensitive to kinase or phosphatase inhibition. Because the amount of surface alpha7 receptors was not affected by kinase or phosphatase inhibitors, these data show that functional properties of alpha7 nAChRs depend on the tyrosine phosphorylation status of the receptor and are the result of a balance between SFKs and tyrosine phosphatases. These findings reveal novel regulatory mechanisms that may help to understand nicotinic receptor-dependent plasticity, addiction, and pathology.

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Tyrosine phosphorylation of nicotinic acetylcholine receptor mediates Grb2 binding.

Publications

Tyrosine phosphorylation of nicotinic acetylcholine receptor mediates Grb2 binding.

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