Binding of the nicotinic acetylcholine receptor to SH2 domains of Fyn and Fyk protein tyrosine kinases.

S L Swope,R L Huganir

doi:10.1016/s0021-9258(18)43954-3

Abstract

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.

Highlights

Binding of the acetylcholine receptor (AChR) to theSH2 Domains of Fyn and FykUsing glutathione S-transferasefusion proteins as affinity reagents, we investigated whether thepreviously reported coimmunoprecipitation of the AChR withFyn and Fykwas
Upon incubation of solubilized Torpedo electric organ postsynaptic membrane proteins representing between 1and 30 pg of protein with the affinity reagents, we found that theAChR bound to the Fyn and Fyk
SH2 domain while up to 28% bound to the Fyk SH2 domain when a high concentration of fusion protein (50 pg) wasused

Summary

Coimmunoprecipitation of the AChR and AChR Subunits with Fyn

Binding of AChR to SH2 Domains of Fyn and Fyk scribed above, the postsynaptic membrane proteins were incubated in denaturation buffer containin5g0 mM Tris, pH 7.4,150mM NaCl, 2 mM EGTA, 1 mM EDTA, 1 mM Na,VO, without(nativeprotein)orwith (denatured protein)2% SDS as described above. After 10 min a t room temperature, the membrane proteins were diluted in fusion protein binding buffer taofinal concentrationof 0.3mg of proteidml, 2% Triton X-100, plus or minus 0.4% SDS, incubated on ice for 15 min, and centrifuged a s described above. The solubilized postsynaptic membrane proteins were incubated with preimmune, anti-Fyn, or anti-Fyk antibodies followed by protein A-Sepharoseas previously described (Swope and Huganir, 1993).The immunoprecipitates were washed three times with fusion protein binding buffer and two times wTirtihslEDTAIEGTA buffer.

FY K

To further quantitate thbeinding of the AChR to theFyn and

Dm ENATURED

DISCUSSION

These kinetic experiments indicated apparent Kd values of