Mammalian EGF receptor activation by the rhomboid protease RHBDL2

Abstract

The epidermal growth factor receptor (EGFR) has several functions in mammalian development and disease, particularly cancer. Most EGF ligands are synthesized as membrane-tethered precursors, and their proteolytic release activates signalling. In Drosophila, rhomboid intramembrane proteases catalyse the release of EGF-family ligands; however, in mammals this seems to be primarily achieved by ADAM-family metalloproteases. We report here that EGF is an efficient substrate of the mammalian rhomboid RHBDL2. RHBDL2 cleaves EGF just outside its transmembrane domain, thereby facilitating its secretion and triggering activation of the EGFR. We have identified endogenous RHBDL2 activity in several tumour cell lines.

Figure 1

Epidermal growth factor cleavage by RHBDL2. (A) EGF domain structure and position of the Myc tag. Red arrow: primary cleavage event by rhomboid or metalloproteases; grey arrow: putative amino-terminal trimming by metalloproteases. (B–E) Secretion assay in COS7 cells transfected with rhomboids plus (B,C) Myc-tagged or (D,E) untagged EGF. (B) Western blots show that RHBDL2 (R2), but not other mammalian rhomboids (R1, R3, R4), can cleave EGF. Expression of HA-tagged rhomboids is shown below. (C) A serine to alanine (SA) catalytic mutant of RHBDL2 does not cleave EGF. (D) Untagged EGF is a substrate for RHBDL2. The faint bands in lanes 2, 3, 5 and 6 are background EGF cleavage caused by incomplete ADAM inhibition. (E) Secreted EGF cleaved by RHBDL2 runs more slowly than metalloprotease-cleaved EGF. Samples were resolved on an 8% SDS-tris-glycine gel. EGF, epidermal growth factor; HA, haemagglutinin; RHBDL, rhomboid-like 2; WT, wild type.

Figure 2

Colocalization of green fluorescent protein (GFP)–RHBDL2 and epidermal growth factor (EGF). A representative × 40 confocal image is shown.

Figure 3

Identification of the cleavage site in EGF. (A) Western blot showing intracellular cleavage of Spitz and its recognition motif mutants by RHBDL2–KDEL in COS7 cells. HA–RHBDL2–KDEL expression is shown below. (B) In vitro cleavage of an MBP-EGF-Thioredoxin chimera by WT or SA mutant AarA. A schematic representation of the chimera is included. (C) The neo-amino-termini of AarA and GlpG-cleaved EGF, identified by N-terminal sequencing. The P1 residue Ala 1031 is shown in red, P4 and P2′ residues are shown in blue, and the TMD is underlined. Two other putative recognition motifs are indicated. (D) Mutation of the P1 residue (Ala 1031) blocks EGF secretion. FLAG-prolactin and GFP are secretion and transfection controls. (E) Combined mutations of the P4 (Leu 1028) and P2′ (Tyr 1033) residues in EGF impair its RHBDL2-induced secretion. EGF, epidermal growth factor; GFP, green fluorescent protein; HA, haemagglutinin; MBP, myelin basic protein; RHBDL2, rhomboid-like 2; SA, serine to alanine; TMD, transmembrane domain; WT, wild type.

Figure 4

Endogenous RHBDL2 expression and activity. (A) RHBDL2 messenger RNA expression in tumour cell lines assessed by quantitative PCR, expressed relative to a control gene for TBP. The mean±s.d. from three replicate experiments is shown. (B) EGF secretion in cell lines that express little or no RHBDL2 is blocked by BB94. (C) Metalloprotease-independent EGF secretion in cells that have high levels of endogenous RHBDL2. The cell lines shown in the first ten lanes were transduced with EGF lentivirus. EGF secretion±BB94 was determined by immunoblotting. The last two lanes are a positive control for the mobility of RHBDL2-cleaved EGF and represent supernatants from COS7 cells transfected with EGF without RHBDL2, minus BB94 (second last lane) or with RHBDL2 plus BB94 (last lane). (D) Impaired metalloprotease-independent secretion of EGF recognition motif mutants in HeLa cells, which express endogenous RHBDL2. (E) Impaired EGF secretion on RNA interference of RHBDL2 in EGF-expressing HeLa cells, which were transduced with shRNAs for vector, GFP or four non-overlapping RHBDL2-specific shRNA lentiviruses. The percentage knockdown of RHBDL2 mRNA relative to the vector control is indicated. EGF, epidermal growth factor; GFP, green fluorescent protein; RHBDL2, rhomboid-like 2; shRNA, short-hairpin RNA; TBP, TATA box-binding protein; WT, wild type.

Figure 5

EGFR activation by RHBDL2. HEK 293T cells were transfected with (A) untagged EGF (125, 250, 500, 750 and 1,000 ng) plus 50 ng of RHBDL2 or (B) 250 ng of EGF plus the indicated amounts of RHBDL2 WT or SA mutant. All treatments were performed in the presence of 10-μM BB94. Cell supernatants were assayed on A431 cells. The upper panels are anti-EGF western blots of the culture supernatants. The middle and lower panels are A431 cell lysates probed for phospho- or total EGFR, respectively. (C) HEK 293T cells were transfected with EGF±RHBDL2. Supernatants were generated with ±BB94 and assayed on A431 cells. EGFR, epidermal growth factor receptor; HEK, human embryonic kidney; RHBDL2, rhomboid-like 2; SA, serine to alanine; WT, wild type.