Figure 4

ICD modifications affect protease accessibility of the CD44 and NRG1 ectodomains.

Ectodomains of CD44 wt or of uncleavable CD44 KR-Mt mutant show different protease accessibility to trypsin (A) or soluble ADAM10 (B). (C) Merlin regulates trypsin access to CD44 ectodomain. Constitutively active Merlin S518A blocks trypsin access to CD44 (right panel) when compared to wt merlin (left panel). (D) NRG1-S286A reduces NRG1 ectodomain accessibility to chymotrypsin. (E) PKCδ knockdown reduces NRG1 ectodomain accessibility to chymotrypsin. (A) CD44 wt and uncleavable KR-Mt were compared after exposure to increasing trypsin concentrations (37 °C) for 1 hr (upper) and 3 hrs (lower panel). Boxed pairs indicate concentration with largest differences between wt and KR-Mt. Conditions and cleavage detection as in Fig. 1. Relative levels of trypsin-cleaved CD44 shown as inserts. Based on size of single cleavage product, trypsin cuts in the ectodomain stalk region. 64 kDa band most likely represents under-glycosylated CD44. (B) Same as (A) but with soluble ADAM10 (37 °C; concentrations and times indicated). (C) RPM-MC cells were co-transfected with tagged CD44 wt and constitutively active merlin (NF2-S518A). CD44fl and C-terminal cleavage products (see “cleavage” middle panel) were detected as in Fig. 1. Merlin expression was detected by merlin antibody (upper panel). In (C) samples were run on the same gel but several lanes that showed other sample conditions using different trypsin concentrations were removed. (D) HEK293T cells were transfected with NRG1 wt or NRG1S286A, or co-transfected with NRG1 wt and PKCδ shRNA (E). Treatments as indicated. Chymotrypsin cleavage of full-length molecule was quantitated and normalized for tubulin (see column diagrams). Column diagrams show mean values of relative levels ± SD from three independent experiments; ns = non-significant, ***p-value < 0.001, ****p-value < 0.0001. Exemplary immunoblots see Supplemental Fig. 2A,B. Other statistical comparisons not shown: (3D) DMSO vs. TPA-treated NRG1wt: non-significant at 0, 18 and 20 μg/ml, but significant at 22 μg/ml (p-value 0.01) and 24 μg/ml chymotrypsin (p-value 0.0025). DMSO vs. TPA-treated NRG1S286A: non-significant. (3E) DMSO vs. TPA-treated NRG1wt plus scrambled shRNA: non-significant at 0, 18, 20 and 22 μg/ml, but significant at 24 μg/ml chymotrypsin (p-value 0.005). DMSO vs. TPA-treated NRG1wt plus PKCδ shRNA: non-significant.