Abstract
We previously proposed that the dopamine D2 receptor-interacting protein S100B binds to a putative S100B-binding motif at residues R233-L240 towards the N-terminus of the 3rd cytoplasmic loop. We used in vitro pull-down assays with FLAG-tagged fragments of the rat D2 receptor third cytoplasmic loop and in vitro-synthesized S100B to evaluate this hypothesis. Our results indicate that the putative S100B-binding motif is neither necessary nor sufficient for strong binding of S100B. Instead, two residues at the junction of the 5th membrane-spanning domain and the cytoplasmic extension of that a-helical domain, K211-I212, are required for robust, calcium-sensitive binding of S100B. This is also the approximate location of previously identified determinants for the binding of arrestin and calmodulin. A D2 receptor mutation converting I212 to phenylalanine has been described in patients with a hyperkinetic movement disorder.
Significance Statement S100B is a small calcium-binding protein that modulates signaling by the dopamine D2 receptor. Our new data suggest that our previous hypothesis about the involvement of an S100B-binding motif is incorrect, and that an important determinant of S100B binding includes a residue that is mutated in patients with a hyperkinetic movement disorder.
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