Fig. 3: KIRREL1 interacts with N-terminal of SAV1 through its intracellular domain (ICD). | Nature Communications

Fig. 3: KIRREL1 interacts with N-terminal of SAV1 through its intracellular domain (ICD).

From: Cell adhesion molecule KIRREL1 is a feedback regulator of Hippo signaling recruiting SAV1 to cell-cell contact sites

Fig. 3

a KIRREL1 interacts with SAV1 in co-immunoprecipitation assay. HEK293T cells were co-transfected with plasmids encoding Myc-KIRREL1-HA and Flag-tagged Hippo pathway components, and cell lysates were subjected to co-immunoprecipitation assay. TCE total cell extract. b Full-length KIRREL1 but not the ΔICD mutant interacts with SAV1 in co-immunoprecipitation assay. c Schematic of full-length and ICD deletion mutants of KIRREL1. d Full-length, ICD Δ1, and ICD Δ2 but not ICD Δ3 mutant of KIRREL1 interacts with SAV1 in co-immunoprecipitation assay. e The C-terminal fragment of KIRREL1 ICD interacts with SAV1. Purified recombinant His-SUMO-tagged KIRREL1 N-terminal fragment of ICD (ICD80—a.a 521–600) or C-terminal fragment of ICD (ICD77—a.a 681–757) was incubated with cell lysates expressing ectopic Flag-tagged SAV1 (left panel) or MST1 (right panel) and subjected to Ni-NTA pull-down. Bottom panel shows Coomassie-stained gel of purified KIRREL1 ICD fragments. f The C-terminal fragment of KIRREL1 ICD directly interacts with SAV1. Purified recombinant His-SUMO-tagged KIRREL1 N-terminal fragment of ICD (ICD80—a.a 521–600) or C-terminal fragment of ICD (ICD77—a.a 681–757) was incubated with purified Flag-SAV1 and subjected to Ni-NTA pull-down assay. Coomassie-stained gel shows purified HIS-SUMO-tagged KIRREL1 ICD fragments. g Overexpression of KIRREL1-induced inhibition of GTIIC-luciferase activity requires the C-terminal fragment of KIRREL1 ICD. Experiment was performed as in Fig. 2e. The data represent mean ± SD, n = 4, error bars denote the SD between four biological replicates; unpaired two-tailed t-test was used to determine the statistical significance, ***p value < 0.001, ns—0.1346. h Schematic of full-length and deletion mutants of SAV1. i N-terminal flexible domain of SAV1 is required and sufficient for binding with KIRREL1. HEK293T cells were transfected with plasmids encoding KIRREL1 and indicated SAV1 mutants and subjected to co-immunoprecipitation assay. Source data for Fig. 3a, b, d, e, f, g, i are provided as Source Data file.

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