Figure 5. PICK1 switch scaffolding mode dependent on intrinsic PDZ affinity. ; (a) Schematic representation of the experimental setup. Both PICK1 WT (blue) and PICK1 A87L (red) are labeled. Exchange is allowed before binding of PICK1 WT/A87L heterodimers. (b) Representative images of heterodimeric binding as a function of concentration. (c–e) Normalized concentration dependent binding to either LKV (solid lines) or LKI (dashed lines), and quantified in both the WT (blue) and A87L (red) channel as function of linear and logarithmic concentration. LKV PICK1 WT/A87L: Kd*=24 ± 21 nM, Bmax: 51 ± 2%. LKI PICK1 WT/A87L Kd*=123 ± 21 nM, Bmax: 56 ± 2% (see Tables 1–3 for explanation of Bmax values) (n = 3), (****p≤0.0001). (f) Schematic summary of conclusions made from figures c-e. Fitted Bmax values are shown on the y-axis, for PICK1 WT/WT and PICK1 WT/A87L to SCMS expressing LKV and LKI together with avidity and proposed binding mode (g–h) Flp-In T-REx 293 eYFP-PICK1 cells transiently expressing LKV, LKI, LKA or LKV +A with (black bars;+Tet) and without (white bars; −Tet) tetracycline-induced expression of eYFP-PICK1 were surface-labeled with anti-FLAG M1 antibody prior to stimulation of internalization with agonist (10 μM Isoproteronol for 20 min). Subsequently, cells were treated with the antagonist (10 μM Alprenolol, 60 min) to allow recycling to the plasma membrane. Surface receptor immunoreactivity was determined by surface ELISA. Internalization (g) refers to the fractional reduction of surface receptor in response to 25 min of agonist exposure compared with non-treated cells (100%). Recycling (h) refers to the fractional recovery of surface receptor following antagonist incubation for 1 hr. These values were all normalized to the respective signal of non-interacting β2-LKV + A, with and without tetracycline induction, respectively. Data are means ±S.E, n = 6. (***p0.05, One way ANOVA, Bonferroni’s post test compared to LKV +A). Scale bars 10 μm.

Figure 5. PICK1 switch scaffolding mode dependent on intrinsic PDZ affinity. ; (a) Schematic representation of the experimental setup. Both PICK1 WT (blue) and PICK1 A87L (red) are labeled. Exchange is allowed before binding of PICK1 WT/A87L heterodimers. (b) Representative images of heterodimeric binding as a function of concentration. (c–e) Normalized concentration dependent binding to either LKV (solid lines) or LKI (dashed lines), and quantified in both the WT (blue) and A87L (red) channel as function of linear and logarithmic concentration. LKV PICK1 WT/A87L: Kd*=24 ± 21 nM, Bmax: 51 ± 2%. LKI PICK1 WT/A87L Kd*=123 ± 21 nM, Bmax: 56 ± 2% (see Tables 1–3 for explanation of Bmax values) (n = 3), (****p≤0.0001). (f) Schematic summary of conclusions made from figures c-e. Fitted Bmax values are shown on the y-axis, for PICK1 WT/WT and PICK1 WT/A87L to SCMS expressing LKV and LKI together with avidity and proposed binding mode (g–h) Flp-In T-REx 293 eYFP-PICK1 cells transiently expressing LKV, LKI, LKA or LKV +A with (black bars;+Tet) and without (white bars; −Tet) tetracycline-induced expression of eYFP-PICK1 were surface-labeled with anti-FLAG M1 antibody prior to stimulation of internalization with agonist (10 μM Isoproteronol for 20 min). Subsequently, cells were treated with the antagonist (10 μM Alprenolol, 60 min) to allow recycling to the plasma membrane. Surface receptor immunoreactivity was determined by surface ELISA. Internalization (g) refers to the fractional reduction of surface receptor in response to 25 min of agonist exposure compared with non-treated cells (100%). Recycling (h) refers to the fractional recovery of surface receptor following antagonist incubation for 1 hr. These values were all normalized to the respective signal of non-interacting β2-LKV + A, with and without tetracycline induction, respectively. Data are means ±S.E, n = 6. (***p0.05, One way ANOVA, Bonferroni’s post test compared to LKV +A). Scale bars 10 μm.