Membrane Localization is Critical for Activation of the PICK1 BAR Domain

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Abstract
The PSD‐95/Discs‐large/ZO‐1 homology (PDZ) domain protein, protein interacting with C kinase 1 (PICK1) contains a C‐terminal Bin/amphiphysin/Rvs (BAR) domain mediating recognition of curved membranes; however, the molecular mechanisms controlling the activity of this domain are poorly understood. In agreement with negative regulation of the BAR domain by the N‐terminal PDZ domain, PICK1 distributed evenly in the cytoplasm, whereas truncation of the PDZ domain caused BAR domain‐dependent redistribution to clusters colocalizing with markers of recycling endosomal compartments. A similar clustering was observed both upon truncation of a short putative α‐helical segment in the linker between the PDZ and the BAR domains and upon coexpression of PICK1 with a transmembrane PDZ ligand, including the alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor GluR2 subunit, the GluR2 C‐terminus transferred to the single transmembrane protein Tac or the dopamine transporter C‐terminus transferred to Tac. In contrast, transfer of the GluR2 C‐terminus to cyan fluorescent protein, a cytosolic protein, did not elicit BAR domain‐dependent clustering. Instead, localizing PICK1 to the membrane by introducing an N‐terminal myristoylation site produced BAR domain‐dependent, but ligand‐independent, PICK1 clustering. The data support that in the absence of PDZ ligand, the PICK1 BAR domain is inhibited through a PDZ domain‐dependent and linker‐dependent mechanism. Moreover, they suggest that unmasking of the BAR domain’s membrane‐binding capacity is not a consequence of ligand binding to the PDZ domain per se but results from, and coincides with, recruitment of PICK1 to a membrane compartment.