Mechanism of activation of the Drosophila EGF Receptor by the TGF
ligand Gurken during oogenesis
Christian Ghiglione1,*,
Erika A. Bach1,
Yolande Paraiso*,
Kermit L. Carraway, III3,
,
Stéphane Noselli* and
Norbert Perrimon1,2,
1 Department of Genetics, Medical Institute, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA
2 Howard Hughes Medical Institute, Harvard Medical School, 200 Longwood Avenue. Boston, MA 02115, USA
3 Division of Signal Transduction. Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215
* Present address: Centre de Biochimie. UMR6543/CNRS. Faculté des Sciences. 06108 Nice, France
Present address: UC Davis Cancer Center, 4645 2nd Avenue, Sacramento, CA 95817, USA
View larger version (99K):
[in a new window]
|
Fig. 1. mbGrk and secGrk overexpression in somatic tissues lead to Egfr activation. (A) Structure of mbGrk and secGrk. SP, signal peptide; EGF, EGF motif; TM, transmembrane domain; C, cytoplasmic domain. The arrow represents the site where the stop codon was introduced to generate secGrk. (B-J) Phenotypes observed after UAS-mbGrk and UAS-secGrk ectopic expression in somatic tissues. (B) Wild-type egg with its two dorsal appendages. (C) UAS-mbGrk/CY2: overexpression of mbGrk in all the follicular epithelium leads to a weak eggshell dorsalization (78% of the eggs, n=352). (D) UAS-secGrk/CY2: overexpression of secGrk leads to a very strong dorsalized eggshell phenotype (85% of the eggs, n=244). (E-G) Embryonic cuticles from: wild-type (E), UAS-mbGrk/CY2 (F) and UAS-secGrk/CY2 (G) females. (H) Wild-type wing with five longitudinal veins and two crossveins. (I,J) Weak and strong wing vein phenotypes associated with UAS-mbGrk/MS1096 (I) and UAS-secGrk/MS1096 wings (J).
|
|
View larger version (44K):
[in a new window]
|
Fig. 2. UAS-mbGrk and UAS-secGrk overexpression in the oocyte leads to Egfr activation. (A) 100% (n=122) of the eggs derived from UASp-mbGrk/pCOG females exhibit a strong dorsalized eggshell. (B) Only 11% (n=441) of the eggs derived from UASp-secGrk/pCOG females exhibit a weak dorsalized eggshell.
|
|
View larger version (116K):
[in a new window]
|
Fig. 3. mbGrk and secGrk bind Egfr, but only secGrk triggers signaling. (A-C) Aggregation of mbGrk-expressing cells with Egfr cells. Sf9 insect cells were independently infected with wild-type baculovirus, or virus encoding Egfr, mbGrk or Kek1. The two populations of cells were mixed and incubated together for 1 hour and then examined. (A) Cells expressing mbGrk strongly aggregate to cells expressing Egfr. (B) Cells expressing mbGrk and cells expressing Kek1 do not aggregate. (C) Cells expressing mbGrk and cells expressing Egfr, in presence of conditioned medium from cells expressing secGrk, no longer aggregate. (D) secGrk but not mbGrk activates Egfr in vitro. S2:Egfr cells (2x106) were incubated for 3 hours with 60 µM CuSO4 to induce moderate levels of Egfr expression (Schweitzer et al., 1995a). The cells were then stimulated for 30 minutes with serum free media (SFM), mbGrk, secGrk or secSpi. Egfr precipitates were prepared using rabbit anti-Egfr polyclonal sera. Top: the level of tyrosine phosphorylation of the Egfr was assessed by western blotting with mAb RC-20 (PY). Bottom: Egfr expression was then assessed by western blotting with rabbit anti-Egfr polyclonal sera. The lower amounts of Egfr in secGrk and secSpi lanes are most probably due to incomplete stripping of the RC-20 antibody.
|
|
View larger version (49K):
[in a new window]
|
Fig. 4. The extracellular domain of Grk is released from the oocyte and internalized in follicle cells. (A) Structure of mbGrkmyc. (B) All eggs laid by UASp-mbGrkmyc/nos-Gal4 females have a strongly dorsalized eggshell. (C,D) Confocal sections showing UASp-mbGrkmyc/nos-Gal4 egg chambers stained with an anti-Grk antibody that detects the N-terminal region of the protein (C), or with an anti-Myc antibody that recognizes the intracellular region of the protein (D). While the uptake of the extracellular domain of Grk is clearly visible in follicle cells (C), the intracellular domain of Grk remains confined to the oocyte (D). Grk and Myc are in green, and actin is in red. (E) Western blot probed with anti-Myc antibody reveals a cleavage of mbGrkmyc. Lane 2 is a protein lysate from UASp-mbGrkmyc/nos-Gal4 ovaries in which two new bands are detected (arrows). The higher band corresponds to the full-length mbGrkmyc protein whereas the lower one corresponds to a cleaved form. These two bands are absent in extracts from wild-type ovaries (lane 1).
|
|
View larger version (102K):
[in a new window]
|
Fig. 6. Synergy between mbGrk, Star and Rho-related proteins during oogenesis. (A) Reduction of S by expressing an antisense UASp-S in the germline causes the ventralization of the eggshell (88%, n=225). (B) Dorsal appendages are fused in S mutant follicle cell clones. (C) Misexpression of UAS-mbGrk in S/+ follicle cells results in a complete suppression of the mbGrk phenotype (compare with Fig. 1C). (D) Co-expression of UAS-S and UAS-mbGrk with CY2-Gal4 causes a strong eggshell dorsalization phenotype. (E) Ectopic expression of UAS-brho in the follicle cells using CY2-Gal4 causes a weak dorsalized eggshell phenotype. (F) Co-expression of UAS-mbGrk and UAS-brho in follicle cells induces a strong dorsalization of the eggshell. (G) Strong dorsalization of the eggshell after overexpression of mbSpi in the oocyte using pCOG-Gal4. (H) Ectopic expression of secSpi in the oocyte using the same driver causes a weaker dorsalized eggshell phenotype (33% of the eggs, n=167).
|
|
View larger version (66K):
[in a new window]
|
Fig. 7. S and Rho-1/Brho in S2 cells. S2 cells were transiently transfected with actin5c (Ac5c) driving expression of the indicated constructs. 72 hours after transfection, cellular lysates and conditioned media were analyzed by western blot with antibody raised against Grk extracellular domain. mbGrkmyc was used to distinguish easily the membrane-bound Grk and cleaved Grk forms. (A) Cell lysates: secGrk proteins exhibited Mr of about 40-45 kDa (lane 2), while mbGrkmyc displayed Mr around 70 kDa (lane 3). Lysates from S2 cells expressing mbGrkmyc with Rho-1 or BrhoGFP (lanes 6 and 7); or mbGrkmyc with Rho-1 or (BrhoGFP) and Smyc (lanes 4 and 5) contained both the mbGrkmyc form and also a cleaved form of Grk that migrated with the secGrk species (lane 2). Lysates from cells expressing mbGrkmyc with Smyc contained only the larger mbGrkmyc species and not the cleaved form of Grk (lane 8). (B) Immunoprecipitates of conditioned media from the transfections described in A: no Grk was detected in media from cells expressing empty vector (lane 1), mbGrkmyc alone (lane 5), mbGrkmyc with Rho-1 or BrhoGFP (lanes 8 and 9, respectively), mbGrkmyc with Smyc (lane 10). The co-expression of mbGrkmyc with both S and Rho-1/BrhoGFP (lanes 6 and 7, respectively) leads to the accumulation of a soluble cleaved form of Grk in the medium that migrates with secGrk (lane 4). As control, secGrk and mbGrkmyc lysates were loaded on the same gel (lanes 2 and 3, respectively). We note that cleaved Grk released in the medium is slightly higher that the engineered secGrk. (C) S and Brho intracellular localization: S2 cells were transfected with Ac5c-BrhoGFP (C1-3) or Ac5c-Smyc (C4-6), both shown in green. The cells were also stained (all shown in red) with phalloidin to mark the plasma membrane (C1 and C4), anti-Drosophila Golgi mAb (C2 and C5) or anti-KDEL to mark the ER (C3 and C6). Brho is localized in discrete vesicles that colocalize with the Golgi apparatus (C2) and not the ER (C3). S is mostly expressed in a peri-plasma membrane pattern (C4). We note that some of S colocalizes with the ER (C6), however, none co-localizes with the Golgi (C5).
|
|
© The Company of Biologists Ltd 2002
19AAmyc. (B) 7% (n=334) of the eggs laid by UASp-mbGrk