Rhomboid and Star facilitate presentation and processing of the Drosophila TGF-alpha homolog Spitz

Abstract

Activation of the Drosophila epidermal growth factor receptor (DER) by the transmembrane ligand, Spitz (Spi), requires two additional transmembrane proteins, Rhomboid and Star. Genetic evidence suggests that Rhomboid and Star facilitate DER signaling by processing membrane-bound Spi (mSpi) to an active, soluble form. To test this model, we use an assay based on Xenopus animal cap explants in which Spi activation of DER is Rhomboid and Star dependent. We show that Spi is on the cell surface but is kept in an inactive state by its cytoplasmic and transmembrane domains; Rhomboid and Star relieve this inhibition, allowing Spi to signal. We show further that Spi is likely to be cleaved within its transmembrane domain. However, a mutant form of mSpi that is not cleaved still signals to DER in a Rhomboid and Star-dependent manner. These results suggest strongly that Rhomboid and Star act primarily to present an active form of Spi to DER, leading secondarily to the processing of Spi into a secreted form.

Figure 1

Rhomboid- (Rho) and Star-dependent mSpi activation of DER in Xenopus animal caps. (A) Models for Rhomboid and Star action. (B) RPA showing that Rhomboid and Star each weakly promote mSpi activation of DER, however, together they are synergistic (lanes 2–5). sSpi activates DER in the absence of Rhomboid and Star (lane 1). Argos represses sSpi activation of DER (lane 7). (C) Induction of XBra expression by DER is reduced in the presence of the dominant-negative FGF receptor, XFD (compare lane 1 with 3, and lane 2 with 5). In a positive control for the potency of XFD, lanes 6 and 7 show that XFD abolishes induction of XBra by activin. (D) Sandwich assay experimental design. (E) RPA of animal cap sandwiches showing that Rhomboid and Star must be coexpressed with the ligand, and not the receptor, for activation to occur (lanes 2–4). When Rhomboid and Star are present in both the signaling and receiving cells, the level of DER activation is attenuated (lanes 5–7).

Figure 2

Analysis of TGF-α/Spi chimeras. (A) Schematic of TGF-α/Spi chimeric molecules. (B) RPA showing that the human EGFR is activated by human TGF-α (lane 3). Together the mSpi C and TM domains (TGF-α/SpiTMC) confer Rhomboid and Star dependence on TGF-α (lanes 4,5). Replacement of the TGF-α C or TM domains with those of mSpi results in constitutive activity (TGF-α/SpiC and TGF-α/SpiTM, lanes 6–9). The observation that TGF-α/SpiC exhibits reduced activity compared with TGF-α could reflect the loss of the TGF-α carboxy-terminal valines, which are normally required for targeting TGF-α to the cell surface (Briley et al. 1997). Spi/TGF-αTMC, in which the mSpi C and TM domains are replaced with those of TGF-α, is Rhomboid and Star independent (lanes 10,11). SpiΔ53C exhibits Rhomboid and Star-independent activity (lanes 15,16).

Figure 3

(A) Western analysis of biotinylated mSpi^myc. mSpi^myc can be biotinylated in the absence of Rhomboid and Star (cf. lanes 3 and 4). mSpi^myc is detected by the anti-human c-myc antibody 9E10 and is indicated by an arrow. (Lanes 1,2) mSpi^myc present in lysates prior to incubation with streptavidin-agarose (10% of the total lysate was loaded). (Lanes 3,4) mSpi^myc eluted from streptavidin–agarose (the entire eluted fraction was loaded). (B) mSpi titration. Similar levels of DER activation are obtained over a range of 500 pg to 1 pg of injected mSpi RNA. (C) Experimental design for Rhomboid and Star-dependent production of Spi conditioned medium (CM). (D) RPA analysis of CM activities. sSpi CM activates DER independently of Rhomboid and Star (lane 1). mSpi CM induces XBra expression in DER-injected animal caps in a Rhomboid and Star-dependent manner (lanes 2,3), but not in uninjected animal caps (ectoderm, lanes 4–6). CM from animal caps expressing Spi-15aa, Rhomboid, and Star does not activate DER, whereas the positive control CM from animal caps expressing mSpi, Rhomboid, and Star efficiently activates DER (cf. lanes 7 and 8). (E) Spi-15aa strongly activates DER in a Rhomboid and Star-dependent manner, similar to mSpi.

Figure 4

Analysis of Spi/NICD chimeras. (A) Spi/NICD chimeric molecules. (B) RPA showing that Spi/NICD and Spi-15aa/NICD activate DER in a Rhomboid and Star-dependent manner, similar to mSpi (lanes 1–6). (C) RPA showing that NICD induces Esr-1 expression (lane 2); however, the Spi/NICD chimeric molecule only activates Esr-1 when Rhomboid and Star are present (lanes 3,4). Spi-15aa/NICD does not activate Esr-1 (lanes 5,6). We note, induction of Esr-1 and XBra expression by Spi/NICD were analyzed as separate experimental samples because together XBra and noggin synergize to promote dorsal mesoderm formation, a background in which Esr-1 is poorly induced (Cunliffe and Smith 1994). (D) Model for Rhomboid and Star-dependent activation of Xbra and Esr-1 by the Spi/NICD chimera. Rhomboid and Star present Spi/NICD, and then cleavage releases both Spi and NICD to activate their respective targets. However, Spi does not need to be cleaved to be active.

Figure 5

Model: mSpi is on the cell surface but it is inactive. Rhomboid and Star present an active form of mSpi, leading to, but not requiring, cleavage of its extracellular domain.