Table 1.
Protein interactions with the AChR
| Protein | Wild-type cells1-a | Rapsyn −/− cells | |||||
|---|---|---|---|---|---|---|---|
| Mild extraction | Stringent extraction | Mild extraction | |||||
| −Agrin | +Agrin | Efficiencyb(%) | −Agrin | +Agrin | −Agrin | +Agrin | |
| MuSK | + | ++ | 1.94 ± 0.13 | + | + | + | + |
| Rapsyn | + | + | 7.58 ± 1.94 | + | + | N/A | N/A |
| src-related kinases | + | + | 0.35 ± 0.07 | + | + | + | + |
| Utrophin1-c | + | + | 0.48 ± 0.17 | + | + | − | − |
| β-dystroglycan1-c | + | + | 0.30 ± 0.04 | − | − | − | − |
| Syntrophin | − | − | 0 | ND | ND | ND | ND |
| Transferrin receptor | − | − | 0 | ND | ND | ND | ND |
Data were obtained by incubating C2, rapsyn wild-type, or rapsyn −/− myotubes with or without 5 nm neural agrin for 16 hr. Cell lysates were prepared in a buffer containing 1% digitonin and 50 mm NaCl (mild conditions) or 1% Triton X-100, 0.5% deoxycholate, and 470 mm NaCl (stringent conditions), as detailed in Materials and Methods. AChRs were isolated using α-bungarotoxin-Sepharose, and associated proteins were identified by immunoblotting. ND, Not done; N/A, not applicable.
As wild-type cells, C2 and rapsyn wild-type myotubes (12-10) were used.
The efficiency of the interaction was calculated for untreated C2 myotubes. It represents the percentage of the total protein associated with total AChR, based on the observation that 14.9% of AChRs in a cell extract can be isolated on α-BT-Sepharose (Fuhrer et al., 1997). Data represent mean ± SD of at least five experiments.
For utrophin and β-dystroglycan, substantial non-specific binding occurs to the Sepharose resin.