Fig. 1.

Interaction between α7 nAChR and PICK1 in yeast. (A) Yeast strain AH109 was cotransformed with plasmids encoding the GAL4 DNA-binding domain fused to different sequences of the cytoplasmic loop of rat α7 nAChR (or rat α4or β2 nAChR, as indicated) and the GAL4 activation domain fused to different PICK1 sequences. Protein–protein interaction was assayed by growing the yeast on selective medium and by galactosidase assays. The specificity of this interaction was tested using control plasmids; + indicates interaction, – no interaction. n.d., not done. (B) PICK1 prey constructs used. CC, coiled coil domain; AR, acidic region. (C) Sequence alignment of the bait 9 region of α7 with other nAChR subunits and with the C-terminus of other proteins known to bind PICK1. A separate alignment of α7 with Arf1 and Arf3 is shown at the bottom. Note the two putative PDZ-binding motifs, EVRYand ESEV. (D) Mutation of the putative PDZ-binding motifs (EVRY and ESEV) in α7 nAChR bait 1 and bait 9. The interaction with PICK1 prey vectors was not affected. Polarity colors mark the residues according to the polarity of amino acids (www.clcbio.com).