Binding of the nicotinic acetylcholine receptor to SH2 domains of Fyn and Fyk protein tyrosine kinases.
The nicotinic acetylcholine receptor (AChR) is phosphorylated on tyrosine both in vitro and in vivo. To identify the protein tyrosine kinase that phosphorylates the receptor, we have previously cloned and characterized two protein tyrosine kinases, Fyn and Fyk, that are highly expressed in Torpedo electric organ, a tissue enriched in the AChR. Both kinases were shown by coimmunoprecipitation to be specifically associated with the AChR. In this study, we examined the molecular basis for the interaction of Fyn and Fyk with the AChR using fusion proteins containing the SH2 domains of the two kinases as affinity reagents. The AChR bound specifically and in a protein concentration-dependent manner to the SH2 domain fusion proteins of Fyn and Fyk. Quantitation of the association revealed that the binding of the AChR to Fyn and Fyk SH2 domain fusion proteins was to a single class of saturable high affinity sites. In addition, the association of the AChR with the SH2 domain fusion proteins was dependent on tyrosine phosphorylation of the AChR and was mediated by the delta subunit of the receptor. Furthermore, upon dissociation of the AChR into subunits, the delta subunit coimmunoprecipitated with both Fyn and Fyk. These data suggest that the association of the AChR with Fyn and Fyk is mediated by an interaction of the tyrosine-phosphorylated delta subunit of the receptor with the SH2 domains of the protein tyrosine kinases.
Department of Neuroscience, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
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Huganir
D000595:Amino Acid Sequence; D000818:Animals; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D011978:Receptors, Nicotinic / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014101:Torpedo
D011518:Proto-Oncogene Proteins; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins; C084253:fyk kinase; D011505:Protein-Tyrosine Kinases; D051076:Proto-Oncogene Proteins c-fyn