Extended Data Figure 4. Mapping of the FBXL2-binding domain in IP3R3, and evidence that the N-terminal suppressor domain inhibits the IP3R3-FBXL2 interaction.

a, Schematic representation of IP3R3 mutants. Binding of IP3R3 to FBXL2 is indicated with the symbol (+). b, HEK293T cells were transfected with GFP-tagged FBXL2 and the indicated Flag-tagged IP3R3 truncated mutants. Whole-cell lysates (WCL) were immunoprecipitated (IP) with anti-Flag resin and proteins were immunoblotted as indicated. This experiment was performed twice. c, HEK293T cells were transfected with GFP-tagged FBXL2 and HA-tagged In IP3R3(1-602) constructs. Sixteen hours after transfection, cells were incubated with MG132 for 3 h before stimulation with ATP for 30 min. Whole-cell lysates were immunoprecipitated (IP) with an anti-HA resin and proteins were immunoblotted as indicated. This experiment was performed twice. d, HeLa cells stably transfected with Flag-tagged FBXL2 under the control of a doxycycline-inducible promoter were treated with doxycycline (0.4 μg ml⁻¹) for 16 h and incubated with or without MG132 (during the last 3 h), in the presence or absence of MRS 2578, an antagonist of P2Y6 receptor (during the last 90 min), as indicated. Cells were subsequently treated with or without ATP for 30 min. Whole-cell lysates were immunoprecipitated (IP) with an anti-Flag resin and proteins were immunoblotted as indicated. Right panel shows quantifications of IP3R3 levels compared to untreated cells (UT), which were set as 100%. P values were calculated by one-way ANOVA and multiple-comparisons test. Error bars indicate s.e.m. The fact (shown in b) that fragments encoding IP3R3(436–587) and IP3R3(227–602) interact with FBXL2 better than the IP3R3(1–602) fragment suggests that the N terminus of IP3R3 inhibits the interaction between FBXL2 and IP3R3. It has been shown that removal of the N-terminal suppressor domain (amino acids 1–226) increases the binding of IP3 to the IP3 binding core domain (amino acids 227–579), and IP3 binding evokes conformational changes that open the suppressor domain^29–32. These conformational changes may allow FBXL2 to access the degron of IP3R3 (that is, the amino acid region required for binding to FBXL2). Thus, in agreement with previous data indicating that IP3 causes the ubiquitination and downregulation of IP3Rs (ref. 27), our results suggest that IP3 promotes the binding of FBXL2 to IP3R3 and that FBXL2 preferentially binds IP3R3 in its open conformation (upon IP3 binding). Accordingly, treatment of cells with ATP, which induces IP3 production and repositioning of the N-terminal suppressor domain, increased the binding between FBXL2 and IP3R3, particularly if proteasomal degradation was inhibited by MG132. This suggests that once IP3 (produced after ATP stimulation) unmasks the IP3R3 degron, FBXL2 binds IP3R3 and this interaction is preserved when FBXL2-mediated degradation of IP3R3 is inhibited. So, ATP and MG132 appear to synergize with each other in promoting and preserving the FBXL2–IP3R3 interaction, respectively. e, Serum-starved (SS) and exponentially (EXP) growing NHFs (passage 2 and 3) were treated with or without ATP as indicated. Cells were subsequently harvested at the indicated time points for immunoblotting. The graph shows the quantification of IP3R3 levels. Error bars indicate s.e.m. f, HEK293T cells were transfected with GFP-tagged FBXL2 and the indicated Flag-tagged IP3R3 deletion mutants (in the context of the 436–587 domain of IP3R3). Whole-cell lysates (WCL) were immunoprecipitated (IP) with anti-Flag resin, and proteins were immunoblotted as indicated. This experiment was performed twice. g, The experiment was performed twice as in f, except that different IP3R3 mutants were used. h, Alignment of the amino acid regions containing the FBXL2-binding motif in IP3R3 orthologues. i, Alignment of the amino acid regions containing the FBXL2-binding motif in human IP3R3 with the corresponding region in IP3R1 and IP3R2. j, HEK293T cells were transfected with the indicated constructs. Whole-cell lysates (WCL) were immunoprecipitated (IP) with anti-Flag resin and immunoblotted as indicated. The bracket on the right marks a ladder of bands corresponding to polyubiquitinated IP3R3. This experiment was performed twice. Unless otherwise noted, experiments were performed at least three times. For gel source data, see Supplementary Fig. 1.