Avidity of Scaffolding Interactions Studied on Inverted Membrane Sheets Using the Synaptic Scaffolding Protein PICK1

Abstract

Maintaining membrane proteins at the right place at the right time is crucial to cellular function. To support such organization a broad spectrum of regulating proteins from the cytosol bind, recruit and arrange the membrane proteins into specific structures. PICK1 is an abundant scaffolding protein interacting with more than 30 different receptors, transporters and ion channels, embedded in lipid membranes. PICK1 contains a single N-terminal PDZ domain and forms homodimers via its central membrane bending and curvature sensing N-BAR domain thus forming a functional unit with two PDZ domains. The PICK1 PDZ domain binds many different PDZ peptide ligands such as GluA2, mGluR7 and the dopamine transporter with no apparent conserved binding motif. The wide span of affinities seen for such proteins raises the question of the importance of the affinity for the isolated domain interactions in context of the overall avidity and the functional effects of these interactions. We have established a system based on the previously published supported membrane sheet system to study the binding of PICK1 to a cell membrane expressed ligand to determine the avidity for “on membrane interactions”. Secondly, we address the functional effects of lowering the affinity of the PDZ domain interactions on the functional effects of PICK1. We demonstrate a dramatic increase in the binding Kd for the oligomeric interaction compared to affinities previously reported in non-native condition binding assays. Furthermore, we show that the interaction is only facilitated by functional PDZ domain. We also observe a significant change in Bmax for lower affinity ligands indicating that the increase of the PDZ affinities might rely on a cooperative binding mechanism of the PICK1 homodimer, which is consistent with results showing that the homodimer binds tighter than the monomer.