Figure 6. Disruption of the PICK AH compromises binding strength and alters synaptic localization and clustering of PICK1 in hippocampal neurons.
(a) PICK1 V121E,L125E amphipathic helix mutations depicted on PICK1 domain overview. (b) Normalized malemide stained PICK1 V121E,L125E binding as a function of concentration towards LKV. Kd*=0.28 ± 0.12 μM, Bmax: 95 ± 9%. Malemide stained PICK1 WT as dashed line for comparison. (c–d) Flp-In T-REx 293 eYFP-PICK1 WT or eYFP-PICK1 V121E, L125E cells transiently expressing LKV with (black bars;+Tet) and without (white bars; −Tet) similar to the data shown in Figure 3h–g. Data are means ± S.E, n = 6. (*p≤0.05, Students t-test, correction for multiple comparisons). (e) Model of PICK1 scaffolding. After the initial binding using one PDZ domain, PICK1 will be able to maintain avidity via both the second PDZ domain and the AH (grey box). The AH interaction, however, is dependent on the PDZ interaction. Under conditions yielding the second interaction relatively unfavorable state (2) will be slightly favored. This will allow for an additional PICK1 molecule to bind the free receptor site. If the concentration and intrinsic affinity is sufficiently high this state (3) can be maintained. (f) Representative images of hippocampal neurons, DIV 20–22, expressing GFP-PICK1WT (upper panel) or GFP-PICK1 V121E, L125E (lower panel) stained for GFP (left column) and PSD-95 (middle column), and the overlay is shown in the right column. Scale bar is 20 µm. (g) Quantification of the expression level of GFP-PICK1 WT and GFP-PICK1 V121E, L125E measured as the mean intensity of GFP in the soma. (h) Quantification of the number of dendritic PICK1 clusters (see Materials and method section for definition) relative to the number of PSD-95 clusters. (i) Quantification of PSD-95 clusters relative to dendritic length. (j) Quantification of the fraction of PICK1 clusters colocalized with PSD-95. All values are normalized to the corresponding mean value for GFP-PICK1 wt. (***p<0.001).