Ca2+/CaM association with the Cobl Homology domain of Cobl promotes Cobl/syndapin I complex formation.
(A) Coimmunoprecipitations of GFP-Cobl1-408/Flag-syndapin I (Flag-Sdp I) and GFP-Cobl1-408∆CaM/Flag-syndapin I complexes from HEK293 cells under Ca2+-free conditions (−), 2 μM Ca2+ (+) and 2 μM Ca2+ with subsequent incubation with EGTA (2 μM Ca2+/1 mM EGTA, [+]). The syndapin I interaction with Cobl is promoted upon activation of Ca2+ signaling (2 μM Ca2+) (indicated by the green upright arrowhead). For further confirmation of the Ca2+/CaM association-dependent increase of the syndapin I interaction, also see coimmunoprecipitations under alternative conditions shown in S12 Fig. (B,C) Quantitative evaluations of coimmunoprecipitated syndapin I normalized to immunoprecipitated Cobl and displayed as mean percental difference ± SEM from the respective Ca2+-free conditions show that the syndapin I interaction is reversibly promoted by increasing calcium. Note that syndapin I coimmunoprecipitation by the CaM binding-deficient Cobl mutant Cobl1-408∆CaM is insensitive to changes of calcium levels. The observed regulation of Cobl/syndapin I complex formation thus requires the CaM binding interface of Cobl. GFP-Cobl1-408/Flag-Sdp I +/− Ca2+, n = 3 each; with Ca2+/EGTA, n = 2. GFP-Cobl1-408∆CaM/Flag-Sdp I +/− Ca2+, n = 3 each; with Ca2+/EGTA, n = 2. Statistical significances were calculated by one-way ANOVA with Tukey’s post-test. *p < 0.05. (D,E) Coimmunoprecipitations from rat brain lysates (D) demonstrate the promotion of endogenous Cobl/syndapin I complexes upon calcium addition (highlighted by green upward arrowhead; E, quantitation; n = 2. For data underlying B, C, and E, see S1 Data.