Figure 1.

NHERF preferentially binds to the motif D-S/T-x-L at the end of target proteins. (A) Binding of the first PDZ domain of NHERF to the β₂ receptor tail is inhibited by point mutations of the final residue of the tail. GST fusion proteins of the β₂ receptor tail with mutations at the terminal position were expressed and purified. Equal amounts (≈25 μg) were loaded on SDS/PAGE gels, blotted, and overlaid with NHERF PDZ1 fusion protein (50 nM). The mutations of the β₂ receptor tail (D-S-L-L) are indicated in bold at the top of the panel. (B) Point mutations at the −1, −2, and −3 positions of the β₂ receptor tail differentially alter NHERF PDZ1 binding. Equal amounts of various β₂ receptor tail point mutants were loaded on an SDS/PAGE gel, blotted, and probed for NHERF PDZ1 fusion protein binding. (C) Summary of NHERF PDZ1-binding preferences at the final four amino acid positions of target proteins based on single amino acid substitution studies of the β₂ receptor tail. The optimal amino acid for each position is shown in the first line of the table. “Good” means that the given change causes less than a twofold loss in NHERF PDZ1 binding relative to the optimal amino acid at that position, whereas “marginal” means that the given change causes more than a twofold but less than a 10-fold decrease in binding. “Poor” means that the given change leads to a >10-fold decrease in NHERF PDZ1 binding. The results are representative of two to four independent determinations for binding to each mutant tail.