Figure 3. FRMD8 binds to the iRhom2 N-terminus.

(A) Schematic representation of truncated human iRhom2 constructs used in (B–E). (B, C) Lysates and anti-HA immunoprecipitation (HA-IP) from HEK293T cells transiently co-transfected with FRMD8-V5 and either empty vector (vect) or truncated human iRhom2-3xHA constructs were immunoblotted for V5 and HA. (D) iRhom1/2 double knockout HEK293T cells stably expressing empty vector (vect) or human iRhom2-3xHA constructs were transiently transfected with alkaline phosphatase (AP)-tagged AREG and then incubated with 200 nM PMA or with DMSO for 30 min. AP activity was measured in supernatants and cell lysates. Each experiment was performed in biological triplicates. The results of three independent shedding experiments are shown. Statistical analysis was performed using a Mann-Whitney test. ****=p value<0.0001. (E) Lysates from iRhom1/2 double knockout HEK293T cells transiently transfected with empty vector (vect) or human iRhom2-3xHA constructs were immunoblotted for ADAM17 and HA.

Figure 3—figure supplement 1. A) Amino acid sequence alignment of human and mouse iRhom2 N-terminal region using Clustal Omega.

The region required for FRMD8 binding is highlighted in red. Conserved phosphorylation sites that have been mutated to alanine in the iRhom2^pDEAD (Figure 10—figure supplement 1) are marked in yellow. Grey residues indicate additional phosphorylation sites that have been reported on PhosphoSitePlus (www.phosphosite.org). An asterisk (*) indicates positions which have a fully conserved residue, a colon (:) indicates strongly similar properties of the amino acids, and a period (.) indicates weakly similar properties according to the Clustal Omega tool. (B) Lysates and anti-HA immunoprecipitation (HA-IP) from HEK293T cells transiently transfected with FRMD8-V5 and either empty vector (vect), mouse iRhom2^WT (WT) or Rhom2^cub (Δ268) were immunoblotted for V5 and HA.