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. 2004 Aug 25;24(34):7491–7502. doi: 10.1523/JNEUROSCI.1255-04.2004

Figure 3.

Figure 3.

nPIST interacts with the stargazin C terminus independent of its PDZ-binding domain. A, Yeast two-hybrid screening with screening with the stargazin C terminus that contains a point mutation in the PDZ-binding domain [stargazin(203-323)T321E] yielded a clone of nPIST containing half of a predicted coiled-coil (CC), linker region between the last CC and PDZ domains, and PDZ and AC (AC) domains. B, Directed yeast two-hybrid interactions with the nPIST prey [nPIST(183-463)] or the PDZ domains of PSD-95 [PSD-95 (PDZ I-III)] and the cytoplasmic tails of NR2A [NR2A(838-1465)], Kv1.4 [Kv1.4(571-656)], and deletions of stargazin revealed that nPIST binds NR2A and that stargazin residues between amino acids 262 and 283 are necessary for nPIST binding, whereas stargazin binding to PSD-95 requires an intact stargazin PDZ-binding domain. C, ClustalW formatted alignments of stargazin(243-283), ccγ3(239-277), ccγ4(289-389), and ccγ8(280-321), showing a highly conserved region within the stargazin nPIST-binding domain (residues 249-277). D, Deletions within the nPIST prey showed binding to the stargazin C terminus (203-323) and the stargazin C terminus with a missing (203-319) or mutated (T321E) PDZ-binding domain occurred between nPIST amino acid residues 211 and 312 and required the first part of the nPIST PDZ domain independent of a traditional PDZ ligand-binding groove, whereas the complete PDZ domain was required for interaction with the cytoplasmic tail of NR2A (residues 838-1465, NR2A).