HOAJ Biology ISSN 2050-0874 Research Open Access Sarcoglycan complex formation is involved in regulation of EGFR signaling during Drosophila eye development Reina Hashimoto1,2, Hideki Yoshida1,3, and Masamitsu Yamaguchi1, 3* Abstract Background: Mutations of some components of the Dystrophin Glycoprotein Complex (DGC) are known to cause Muscular Dystrophy in humans. An innovative approach to the further understanding of pathological mechanisms and physiological roles of the DGC has been recently made using several non-mammalian animal models, using species such as Caenorhabditis elegans, Xenopus laevis, and zebrafish. The mammalian DGC is in most part conserved in Drosophila, with respect to the amino acid sequence. However, detailed comparisons of the characteristic properties of each DGC components between mammals and Drosophila have yet to be made. Methods: In the present study, we analyzed mutual binding ability of Drosophila Sarcoglycans in order to address the possibility of complex formation, a characteristic of the mammalian counterparts. Results: In vitro binding assays showed that Drosophila Sarcoglycans could mutually bind to each other. Moreover, β-Sarcoglycan and δ-Sarcoglycan could directly associate with Epidermal growth factor receptor (EGFR) via the EGF-like consensus sequences of their C-terminus regions. Conclusions: In vitro data in combination with immunohistochemical data for sarcoglycan knockdown fly retinae, we propose that Sarcoglycan complex formation is required for eye development. Notably, the direct binding of β-Sarcoglycan and δ-Sarcoglycan to EGFR might be critical for the proper regulation of EGFR signaling during eye development in Drosophila. Keywords: Sarcoglycan, Drosophila, EGFR, muscular dystrophy, eye development. Background The Dystrophin Glycoprotein Complex (DGC) stabilizes the plasma membrane structure, especially during cell movement, by linking the internal cytoskeleton and the extracellular matrix. It is basically a heterogeneous large complex consisting of various combinations of Dystrophin, Utrophin, Dystroglycan, Sarcoglycans, Syntrophins, Dystrobrevins and Sarcospan in mammals [1]. The six forms of Sarcoglycan (α-,β-,γ-,δ-,ε- and ζ-Sarcoglycan) in mammals are each encoded by independent gene loci [2-7]. Mutations in α-, β-, γ- and δ-sarcoglycan are reported to cause limb girdle muscular dystrophies (LGMD) [8,9]. Elucidation of human and fly genomic sequences has revealed Drosophila melanogaster to possess fewer redundant orthologues of most human DGC components, with the exception of sarcospan, where Drosophila α-Sarcoglycan (dScgα), β-Sarcoglycan (dScgβ), and δ-Sarcoglycan (dScgδ) correspond to α-/ε-Sarcoglycan, β-Sarcoglycan and γ-/δ-/ζ-Sarcoglycan in human, respectively [10,11]. δ-Sarcoglycan deficient mutant flies have been described to show progressive muscle dysfunction, mobility defects and reduced life span [12]. Correspondence: myamaguc@kit.ac.jp 1 Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585, Japan. Full list of Author’s information is available at the end of the article One of the proposed functions of the Sarcoglycan complex is to stabilize DGC itself by interacting with other constitutive proteins [13-16]. The β-/δ-Sarcoglycan core is known to interact with the C-terminus of dystrophin [13]. Biochemical preparations from sarcoglycan mutant muscle have revealed less tightly adherent α-dystroglycan subunits suggesting abnormal interaction in the absence of Sarcoglycan [15-17]. However, from other studies [18], one can suggest that Sarcoglycans might also have non-mechanical functions, for example functionally compensating for integrins in muscle. It has also been reported that sarcoglycans associate with integrins and nNOS, indicating a potential role in signaling transduction. In fact, we reported possible interactions between dScgβ and Epidermal Growth Factor Receptor (EGFR) signaling in Drosophila, from genetic screening and immunohistochemical analyses of dscgβ knockdown pupal retina [19]. Despite relatively conserved homology based on amino acid sequence comparisons [10], concrete evidence confirmed by experimental results of characteristic and functional similarities of DGC components between mammals and Drosophila is still missing. Therefore, in this study, we performed in vitro binding assays of three Drosophila Sarcoglycans, dScgα, dScgβ and dScgδ. In practice, complex formation of Sarcoglycan subunits is necessary to exert its contribution to the biological roles of the DGC in mammals [13,20]. © 2012 Yamaguchi et al; licensee Herbert Publications Ltd. his is an Open Access article distributed under the terms of Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0). his permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Yamaguchi et al. HOAJ Biology 2012, http://www.hoajonline.com/journals/pdf/2050-0874-1-7.pdf It is commonly assumed that defects in a single Sarcoglycan bring about severe impairment of the appropriate assembly. In turn, the generation of aberrant protein aggregation interferes with the normal function [21]. Our in vitro binding assays demonstrated that all the Sarcoglycans in Drosophila are able to bind to each other, suggesting the potential to form a sub-complex, as observed in mammals. In our previous report, over-activation of EGFR signaling was detected in dscgβ knockdown fly retina, presumably due to ectopic expression of rhomboid, encoding the protease that activates positive EGFR ligands [19]. In the present study, we demonstrated that knockdown of other Sarcoglycan family genes, dscgα and dscgδ also induced ectopic expression of rhomboid in the pupal retinae as observed in the knockdown flies of dscgβ. Moreover, in vitro binding assays of recombinant Sarcoglycans and EGFR revealed direct associations via the highly conserved EGF-like consensus sequences present in the C-termini of dScgβ and dScgδ. Collectively from our findings, we propose that: (1) characteristic functions of Sarcoglycans in terms of the complex formation are conserved between Drosophila and mammals; (2) Sarcoglycan complex formation is required for negative control of EGFR signaling; and (3) direct association of dScgβ and dScgδ with EGFR plays a significant role in the regulation of EGFR signaling during Drosophila eye development. Methods Cloning of target genes into the pETDuet-1 vector For co-expression of two target genes in Escherichia coli (E. coli), we used the pETDuet-1 vector which contains two multiple cloning sites (Novagen). 6×His was tagged for one of the expressed proteins while Flag tag was designed for another. All the genes inserted into the pETDuet-1 vector were amplified by PCR using KOD plus (Toyobo). doi: 10.7243/2050-0874-1-7 The primer pairs and the DNA resources for the PCR reactions, and the restriction enzymes used for the cloning into the pETDuet-1 vector are summarized in Table 1. cDNAs containing EST clones, RH11377 and RE40051, were obtained from Berkeley Drosophila Genome Project, and cDNAs derived from an Oregon R adult female were obtained from mRNA with polyA tails by RT-PCR. For the cloning into multiple cloning site 1 (His-tag) of the pETDuet-1 vector, the PCR products obtained from the cDNA resources were directly inserted at the distinct restriction enzyme sites of a blank pETDuet-1 vector. For cloning of Flag-tagged target genes into the multiple cloning site 2 of the pETDuet-1 vector, the initial PCR products from the cDNA resources were first sub-cloned downstream of the flag tag sequence in a modified pUAST vector in order to add a Flag tag. Then, the target genes with flag sequences were amplified by secondary PCR reactions using the sub-cloned pUAST vectors as DNA templates and inserted into the multiple cloning site 2 of the plain pETDuet-1 vectors or vectors carrying His tagged counterpart target genes to obtain the final objective constructs. Notably, no tag was added to Lamin, since a well-working antibody against Lamin was commonly available from the Developmental Studies Hybridoma Bank (DSHB). A 1677 base pair (bp) fragment encoding 1-559 amino acids of Drosophila EGFR PA was chosen for the truncated form of Drosophila EGFR (Egfr1-559), while a 942bp fragment encoding 1-314 amino acids of dScgβ and a 1131bp fragment corresponding to 1-377 amino acids of dScgδ were employed for the EGF consensus sequence deletion forms of dScgβ (dScgβΔEGF-like) and dScgδ (dScgδΔEGF-like), respectively. The obtained protein products concerning Sarcoglycans in this experiment are schematically represented with rectangular bars in Figure 1. All the constructs were verified by nucleotide sequencing before use. Table 1-Cloning into pETDuet vector. All the used primer pairs, restriction enzymes and DNA resources of the generated constructs for in vitro binding assay using pETDuet vector are listed. F’ and R’ respectively represent forward and reverse primers. he sequences of the used restriction enzyme sites are underlined. 2 Yamaguchi et al. HOAJ Biology 2012, http://www.hoajonline.com/journals/pdf/2050-0874-1-7.pdf doi: 10.7243/2050-0874-1-7 TritonX-100) and the obtained lysate was centrifuged at 16,000×g for 5 min at 4oC. Then, while a part of the supernatant was stored as an Input sample, an equivalent volume from the rest of the supernatant containing 5 mg of total protein was applied for reaction with 100μl of Ni-NTA agarose gel (Qiagen). After incubation for 4h at 4oC, the gels were thoroughly washed with Wash buffer and the binding proteins were separated from the gel with Elution buffer (20mM Tris-HCl, pH 8.0 containing 10% glycerol, 0.5M NaCl, 250mM Imidazole, 20mM β-mercaptoethanol, and 0.5% Tween-20, 0.5% TritonX-100). The Input samples and the elution fractions were applied to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western immunoblot analysis. The proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad) after SDS-PAGE, and the blotted membranes were blocked with TBS-T containing 2% blocking reagents for 1h at 25oC followed by incubation with mouse anti-His (Invitrogen) at 1:2,000 dilution, mouse anti-Flag (Sigma) at 1:4,000 dilution, or mouse anti-Lamin (DSHB) at 1:8,000 dilution for 16h at 4 oC. After washing, the membranes were incubated with HRP-conjugated secondary antibodies (GE healthcare) at 1:100,000 dilution for 1h at 25oC. Detection was performed with ECL Western blotting detection reagent (GE healthcare) and images were analyzed with a Lumivision Pro HSII image analyzer (Aisin seiki). Fly stocks Fly stocks were maintained at 25oC on standard food containing 0.7% agar, 5% glucose, and 7% dry yeast. Canton S was used as the wild type. An enhancer trap line carrying the lacZ marker rhoX63 (rhomboid) was obtained from Y. Hiromi, while others carrying lacZ markers spis3547 (spitz) and argos05845 (argos) and rhove-1 were obtained from the Bloomington Drosophila stock center. Establishment of lines carrying GMR-GAL4 was described earlier [22]. Figure 1-he employed constructs and alignment of the conserved EGF-like consensus sequences. (A) Diagrams of the target region of the constructs for the generation of RNAi lies and for the recombinant proteins for in vitro binding assays. Rectangular bars show the obtained recombinant proteins. Gray areas of the bars represent the EGFlike consensus region. (B) Details of amino acid sequences of the conserved EGF-like sequences are highlighted in gray and the essential cysteine and glycine residues in black in panel B. X can be any arbitrary amino acids. In the C-terminus region, dScgβ and dScgδ possess an EGF-like consensus sequence, which is characteristic of EGFR ligands such as human TGFα and Spitz (Drosophila homologue of TGFα). his sequence is also conserved in mammalian Sarcoglycans except α- and ε-Sarcoglycan. he numbers written below the black-highlighted cysteine (C) indicate the amino acid numbers counted from the N-terminus. Establishment of transgenic lies In order to establish transgenic fly lines applicable for knockdown, two independent regions for each sarcoglycan were designed to construct plasmids with hairpin-loop structures. The plasmid constructs were named αi1, αi2 for dscgα knockdown, βi1, βi2 for dscgβ knockdown and δi1, δi2 for dscgδ knockdown, respectively. Notably, since we have already established transgenic flies carrying UASInverted Repeated (IR)-dscgβ whose target region corresponds to βi1 in our previous work [19], we used these original lines for UAS-IR-βi1 in this study. Therefore, concerning dscgβ knockdown, we only generated transgenic flies carrying UAS-IR-dscgβi2 whose target region is not overlapped with UAS-IR-dscgβi1 in this study. The employed target regions are schematically shown in Figure 1 for easy comparison and the cloning information is summarized in detail in Table 2. All the genes inserted into the pWIZ vector were In vitro protein binding assay obtained from EST clones or cDNAs derived from Oregon R adult Expression of the target genes was induced by 1mM isopropyl-β- females by PCR using KOD plus (Toyobo). NheI/ XbaI sites were D-thiogalactopyranoside (Wako) with incubation for 2 h at 37oC employed for insertion of target genes into multiple cloning site 1, in E. coli BL21DE3. The E. coli were then sonicated in Wash buffer while NheI/ EcoRI fragments were used for compatible cloning into (20mM Tris-HCl (pH 8.0) containing 10% glycerol, 0.5M NaCl, 50mM AvrII/ EcoRI sites of multiple cloning site 2. Thus we constructed Imidazole, 20mM β-mercaptoethanol, and 0.5% Tween-20, 0.5% αi1-IR, αi2-IR, βi2-IR, δi1-IR, and δi2-IR plasmids head to head. The 3 Yamaguchi et al. HOAJ Biology 2012, http://www.hoajonline.com/journals/pdf/2050-0874-1-7.pdf doi: 10.7243/2050-0874-1-7 Table 2. Cloning into pWIZ vector. All the constructs for the establishment of the knockdown lies, the used primer pairs, restriction enzymes and DNA resources are listed. F’ and R7 respectively represent forward and reverse primers. he sequences of the used restriction enzyme sites are underlined. constructs were verified by sequencing and then injected into embryos to obtain stable transformant lines carrying UAS-IR-dscgαi1, UAS-IR-dscgαi2, UAS-IR-dscgβi2, UAS-IR-dscgδi1, and UAS-IR-dscgδi2. P element-mediated germ line transformation was carried out as described earlier [23] and F1 transformants were selected on the basis of white eye color rescue [24]. The established transgenic strains are summarized in Table 3. Five, four, eleven, eight and thirteen transgenic strains carrying UAS-IR-dscgαi1, UAS-IR-dscgαi2, UAS-IR-dscgβi2, UAS-IR-dscgδi1, and UAS-IR-dscgδi2 were established, respectively. To drive expression of the double-stranded RNAs of dscgα, dscgβ or dscgδ in animals, we crossed the transgenic flies with the GMR-GAL4 line. Not all the strains showed a similar extent of the rough eye phenotype, probably due to differences in RNAi efficiency, but at least three strains for each construct displayed a consistent rough eye phenotype. Scanning electron microscopy To assess the eye phenotype, adult flies were anesthetized, mounted on stages, and observed under a scanning electron microscope (SEM) VE-7800 (Keyence) in the low vacuum mode. mRNA preparation and semi-quantitative RT-PCR Total RNA was isolated from the third instar larvae by TRIzol (Invitrogen) followed by DNaseI treatment. mRNA was purified using an OligotexTM-dT30 <super> mRNA purification kit (Takara). Then, cDNA was obtained using oligo (dT) primer and PrimeScript® 4 Yamaguchi et al. HOAJ Biology 2012, http://www.hoajonline.com/journals/pdf/2050-0874-1-7.pdf doi: 10.7243/2050-0874-1-7 Table 3.he established transgenic strains. P-element plasmid, the established strains and their chromosome linkages are shown. Figure 2. In vitro binding assays using recombinant Drosophila Sarcoglycans. (A) Diagrams of the obtained constructs for the production of recombinant proteins. he numbers written below the rectangular bars represent the amino acid numbers counted from N-terminus of respective proteins. (B) Western blots of both Input and Elution samples with anti-His or anti-Flag antibodies are shown. Binding between dScgα and dScgβ (I), dScgα and dScgδ (II), dScgβ and dScgδ (III) was tested. Binding between dScgα and lamin was also examined as a negative control to exclude the possibility of non-speciic binding (IV). 5 mg of total protein in the E. coli extracts was applied for a 100 μl volume of the Ni-NTA agarose gel and eluted with 100 μl bufer. 20 μl of each sample was loaded for SDS-PAGE. RTase (Takara). The following primers were used for the gene amplification. : For dScgα, 5’-AGTTCAGGGTCTAAAGTTGCA-3’ eyes were incubated with diluted primary antibodies in PBST and a n d 5’- A A AG C TG CCC ACG TG G AC A AT- 3 ’, f o r d S c g β , 10% normal goat serum for 16 h at 4 oC. Mouse anti-LacZ (1:500) 5’-CACCAGGGACGCAACACG-3’ and 5’-TGCGGAAGACGCGCACAT-3’, (Promega) and rat anti-Elav (1:200) (DSHB) antibodies were used. f o r d S c g δ , 5 ’ - A T T T C G C A T A A A C G A C A C C A A - 3 ’ Eyes were washed three times with PBST and then incubated with a n d 5 ’ - T C T G C A G A C C G T G G A AT C AT C- 3 ’, a n d f o r secondary antibodies labeled with either Alexa546 or Alexa488 r p 4 9 , 5 ’ - A T G G C A A C A A G C G C C A A A C T G - 3 ’ a n d (Invitrogen) at 25oC for 2h. After three washes in PBST, eyes were 5’-TCAGAAGCCCTTCTCCTTGAG-3’. mounted in mounting medium (Bio-Rad) and analyzed by confocal laser scanning microscopy with a Zeiss LSM510. Immunohistochemistry of pupal retinae For immunohistochemistry, staged eyes were dissected, fixed in 4% formaldehyde, and blocked with 10% normal goat serum in PBST (PBS containing 0.15% Triton X-100). After blocking, the Results Drosophila and Mammalian Sarcoglycans Since it is well known that mammalian Sarcoglycans form functional 5 Yamaguchi et al. HOAJ Biology 2012, http://www.hoajonline.com/journals/pdf/2050-0874-1-7.pdf doi: 10.7243/2050-0874-1-7 Figure 3-Rough eye phenotype of the sarcoglycan knockdown lies. Scanning electron micrographs of adult compound eyes. (A)GMR-GAL4/+;+;+ (B)GMR-GAL4/+;UAS-IR-dscgαi1/UAS-IR-dscgαi1;+ (C)GMR-GAL4/+;UAS-IR-dscgαi2/UAS-IR-dscgαi2;+ (D)GMR-GAL4/+;+;UASIR-dscgβi1/UAS-IR-dscgβi1 (E)GMR-GAL4/+;UAS-IR-dscgβi2/UAS-IR-dscgβi2;+ (F)GMR-GAL4/+;+;UAS-IR-dscgδi1/UAS-IR-dscgδi1 (G)GMR-GAL4/ UAS-IR-dscgδi2 (strain8441);+;UAS-IR-dscgδi2(strain8545)/+ (H)GMR-GAL4/+;UAS-IR-dscgαi2/+;+ (I)GMR-GAL4/+;UAS-IR-dscgβi2/+;+ (J)GMRGAL4/+;+;UAS-IR-dscgδi1/+ (K)GMR-GAL4/+;UAS-IR-dscgαi2/UAS-IR-dscgβi2;+ (L)GMR-GAL4/+;UAS-IR-dscgβi2/+;UAS-IR-dscgδi1/+ (M)GMRGAL4/+;UAS-IR-dscgαi2/+;UAS-IR-dscgδi1/+ (N) Reduction of dscgα, dscgβ or dscgδ mRNA levels in larval tissues. rp49 mRNA was used as an internal control. he graph shows the ratio of knockdown ly values to the wild type. Error bars show standard deviations (n=3). mRNAs were prepared from the knockdown lies in which the expression of each sarcoglycan double strand RNAs was induced by Act5C-GAL4. (Cont)Act5C-GAL4/+ (αi1)UAS-IRdscgαi1/+;Act5C-GAL4/+ (αi2)UAS-IR-dscgαi2/+;Act5C-GAL4/+ (βi1)UAS-IR-dscgβi1/Act5C-GAL4 (βi2)UAS-IR-dscgβi2/+;Act5C-GAL4/+ (δi1)UAS-IRdscgδi1/Act5C-GAL4 (δi2)UAS-IR-dscgδi2/Act5C-GAL4 (O) Western blots of extracts from third-instar larvae of control (Cont), βi1 and βi2 knockdown lies were probed with anti-dScgβ and anti-αTubulin antibodies. (Cont)Act5C-GAL4/+ (βi1)UAS-IR-dscgβi1/Act5C-GAL4 (βi2)UAS-IR-dscgβi2/+;Act5CGAL4/+ hetero-tetramer [13,20], we examined the in vitro binding ability between dScgα and dScgβ, dScgα and dScgδ, or dScgβ and dScgδ by exploiting the pETDuet-1 co-expression system of two target genes in E. coli (Figure 1A). In this experiment, the specific binding between His tag and Ni agarose was utilized for precipitation, and co-precipitation of the counterpart protein containing Flag tag was analyzed by Western immunoblotting. As shown in Figure 2 with input sample treated with anti-His and anti-Flag antibodies (Figure 2B, lanes i-iii and vii-ix), we confirmed the presence of all the target proteins whose cDNAs were carried by the distinct pETDuet-1 vectors in the supernatant after sonication of E. coli and centrifugation of protein extracts. Both His-dScgα and Flag-dScgα were detected at 52kDa by Western blotting (Figure 2B-I, II and IV), consistent with the expected molecular weight of dScgα 50.7kDa according to ExPASy (http://us.expasy. org/tools/pi_tool.html). The apparent molecular weights of HisdScgδ and Lamin were 41kDa and 72 kDa (Figure 2B-II, III and IV), respectively. These are also in accordance with the calculated 6 Yamaguchi et al. HOAJ Biology 2012, http://www.hoajonline.com/journals/pdf/2050-0874-1-7.pdf doi: 10.7243/2050-0874-1-7 molecular weights, 42.6kDa for dScgδ and 71.3kDa for Lamin. On the other hand, we found slightly larger apparent molecular weights of Flag-dScgβ (Figure 2B- I and III) and His-dScgβ (Figure 6B-II) at 48.0 kDa than the estimated molecular weight of dScgβ of 38.1kDa. The slightly slower migration of dScgβ on SDS-PAGE is consistent with our previous report [25]. Exogenously over-expressed dScgβ with Flag tag in flies also exhibited a molecular weight of 48.0 kDa [25]. Since both Flag tagged and His tagged dScgβ showed slower migration on SDS-PAGE, this behavior of dScgβ is likely due to intrinsic properties of the dScgβ amino acid sequence. We also ensured that His-tagged target proteins, His-dScgα and His-dScgδ, bind to Ni agarose gels as detected in the elution samples with anti-His antibodies (Figure 2B, lanes iv and vi). Flag-tagged dScgβ was detected by anti-Flag antibodies as co-precipitation products with His-tagged dScgα, indicating binding between dScgα and dScgβ (Figure 2B-I, lane xii). According to the quantification of the visible band by Lumivision Pro HSII image analyzer, approximately 12.4% of Flag-dScgβ in the input sample was eluted with His-dScgα. Similarly, we observed binding between His-dScgδ and FlagdScgα, and between His-dScgδ and Flag-dScgβ (Figure 2B-II and III, lane xii). 12.5% of Flag-dScgα and 31.8% of Flag-dScgβ in the E. coli soluble lysate were eluted with His-dScgδ. On the other hand, Lamin, which shares similarities with Sarcoglycans as both are transmembrane proteins but which is not supposed to interact with any Sarcoglycans considering differences in sub-cellular localization, was undetectable on elution as expected (Figure 2B-IV, lane xii). These data suggest that the observed binding between dScgα and dScgβ, dScgα and dScgδ, and dScgβ and dScgδ was specific in each case (Figure 2B-I, II and III). Collectively, here we suggest that Drosophila Sarcoglycans can form heterologous complexes as with mammalian homologues, supporting the idea that so far identified Drosophila Sarcoglycans share characters with their corresponding mammalian counterparts. Alteration in EGFR signaling in knockdown lies for sarcoglycans In a previous report, we demonstrated that knockdown of dscgβ (βi1) caused severe morphological aberrancy in the adult compound eyes accompanied by ectopic expression of rhomboid (rho) and consequently increased activation of EGFR signaling in pupal retinae as evidenced by phosphorylation of ERK [19]. In the present study, initially in order to address the off-target effects which are frequently encountered with the RNAi technique, we have newly established and examined fly lines with dscgβ knockdown (βi2), whose target region for the inverted repeat sequence has no overlap with the previously tested construct (βi1) (Figure 1A). βi2 knockdown flies by GMR-GAL4 displayed a mild rough eye phenotype by SEM (Figure 3E). Both RT-PCR and Western blot analyses revealed less reduction of mRNA and protein levels of dScgβ in the βi2 knockdown flies than in βi1 knockdown flies (Figure 3N). βi2 knockdown flies showed reduction of mRNA level to 68% and reduction of protein level to 72% of those of the control flies, while βi1 knockdown flies exhibited 89% decrease in mRNA and 80% in protein (Figure 3N). The data could explain the weaker rough eye phenotype in βi2 than βi1 (Figure 3, panels D and E). However, although the extent of Figure 4-Expression of rhomboid in 35-hour APF retinae. Knockdown lies for any sarcoglycans showed ectopic expression of rhomboid in two unknown cells of each ommatidium. Red and green indicate anti-lacZ and anti-Elav signals, respectively. he white arrowheads indicate examples of cells ectopically expressing rhomboid. Although the level of the luorescence difers in each sample, it does not simply relect the diferences in the expression level as this is mainly due to technical diiculties. (A)GMR-GAL4/+;+;+/X63 (B)GMR-GAL4/+;+;UAS-IR-dscgβi1/ X63 (C)GMR-GAL4/+;UAS-IR-dscgβi2/+;X63/+ (D)GMRGAL4/+;UAS-IR-dscgαi1/+;X63/+ (E)GMR-GAL4/+;UAS-IRdscgαi2/+;X63/+ (F)GMR-GAL4/+;+;UAS-IR-dscgδi1/X63 (G) GMR-GAL4/+;+;UAS-IR-dscgδi2/X63 7 Yamaguchi et al. HOAJ Biology 2012, http://www.hoajonline.com/journals/pdf/2050-0874-1-7.pdf doi: 10.7243/2050-0874-1-7 Figure 5-Scanning electron micrographs of adult compound eyes. (A)GMR-GAL4/Y;UAS-IR-dscgαi1; + (B)GMR-GAL4/Y;UAS-IR-dscgαi1; rhove-1/+ (C)GMR-GAL4/Y;UAS-IR-dscgδi1/CyO;+ (D)GMR-GAL4/Y;UAS-IR-dscgδi1/ CyO;rhove-1/+ (E)GMR-GAL4/Y; UAS-IR-dscgαi1;+ (F)GMR-GAL4/Y;UAS-IRdscgαi1;rhove-1/+ (G)GMR-GAL4/Y;UAS-IR-dscgδi1/CyO;+ (H)GMR-GAL4/Y;UASIR-dscgδi1/CyO;rhove-1/+ Figure 7. Expression of Spitz and Argos in 35-hour APF pupal retinae. Red signals indicate expression of spitz (let panels) or argos (right panels), visualized by anti-lacZ antibody staining, whereas green signals indicates antiElav signals in photoreceptor cells. he white arrowheads indicate examples of the two extra cells per each ommatidium, showing expression of spitz (let panels) or argos (right panels) in the control (upper panels) or dscgβ knockdown (lower panels) lies.(let, upper) GMR-GAL4/+;spik05808/+;+ (let, lower) GMR-GAL4/+;spik05808/+;UAS-IR-dscgβi1 strain72)/+ (right, upper) GMRGAL4/+;arg05845/+;+ (right, lower)GMR-GAL4/+;arg05845/+;UAS-IR-dscgβi1 strain72)/+ Figure 6. In vitro binding between Drosophila Sarcoglycans and EGFR. (A) Diagrams of the obtained constructs for the production of recombinant proteins. he numbers written below the rectangular bars represent the amino acid numbers counted from the N-termini of the respective proteins. (B) Western blots of both Input and Elution samples with anti-His or anti-Flag antibodies are shown. Binding between the extracellular soluble form of Drosophila EGFR (Egfr1-559) and variants of Sarcoglycans was examined. 5 mg of total protein in the E.coli extract was applied for a 100 μl volume of the Ni-NTA agarose gel and eluted with 100 μl bufer. 20 μl of each sample was loaded for the SDS-PAGE. While no binding of Egfr1-559 with dScgα was seen (I-lane 12), dScgβ and dScgδ demonstrated binding to Egfr1-559 (II and IV-lane 12). Furthermore, neither dScgβ nor dScgδ, lacking any EGF-like consensus sequence, showed binding to Egfr1-559 (III and V-lane 12), suggesting that the binding between dScgβ and Egfr1-559, dScgδ and Egfr1-559 is mediated by the EGF-like consensus sequence in the C-terminus regions of dscgβ and dScgδ. 8 Yamaguchi et al. HOAJ Biology 2012, http://www.hoajonline.com/journals/pdf/2050-0874-1-7.pdf roughness in the adult compound eyes examined by SEM apparently differed between βi1 and βi2 knockdown flies, immunostaining data of 35 hour After Puparium Formation (APF) pupal retinae showed similar molecular alteration in terms of ectopic expression of rho in both βi1 and βi2 knockdown flies (Figure 4B and C). These phenotypic similarities of βi1 and βi2 knockdown flies ruled out off-target effects. We also investigated knockdown effects of dscgα and dscgδ with GMR-GAL4 in order to determine whether the above-described phenotypes were induced by functional disorder of dScgβ itself or by the defects in the stability of the whole Sarcoglycan complex provoked by a single Sarcoglycan family protein depletion. Also here, two independent fly lines, whose target regions for inverted repeat constructs were not overlapping, have been established and tested to take into account the off-target problem (Figure 1A). RT-PCR data showed 79% reduction of mRNA level in αi1, and 61%, 81%, and 69%, respectively in αi2, δi1 and δi2 knockdown flies compared to control flies. Although knockdown flies carrying one copy of distinct transgenes (αi1, αi2, δi1 and δi2) showed no obvious rough eye phenotype (Figure 3H-J), an enhanced rough eye phenotype was observed in the knockdown flies with two copies of each transgene (Figure 3B, C, F and G). In addition, synergistic enhancement was also evident in the double knockdown flies carrying inverted repeat sequences of two different sarcoglycans (Figure 3H-M). All the combinations of Sarcoglycans, dScgα-dScgβ, dScgβ-dScgδ and dScgα-dScgδ exhibited an enhanced rough eye phenotype. Furthermore, we detected ectopic expression of rhomboid gene in the 35-hour APF pupal retinae of both dscgα and dscgδ knockdown flies as well as in dscgβ knockdown flies (Figure 4B-G). In addition, half dose reduction of the rho-1 gene significantly suppressed the rough eye phenotype induced by knockdown of dscgα or dscgδ (Figure 5), as observed with the dscgβ knockdown [19]. These results suggest that all three Sarcoglycans, dScgα, dScgβ and Scgδ are necessary for proper level of EGFR signaling to form normal compound eyes in adults. Interactive mechanism of the Sarcoglycan complex and EGFR signaling Although we indicated that Sarcoglycan complex could negatively regulate EGFR signaling in a physiological state [19], we still had no idea of how Sarcoglycan complex affects activation or inactivation of EGFR signaling. In order to address whether it is conducted by direct interaction with EGFR signaling molecules or via an indirect pathway, we performed an immunohistochemical expression pattern analysis of the major components in the EGFR signaling pathway like rho (Figure 4). In pupal retinae, only rhomboid showed different expression patterns between control and dscgβ knockdown flies (βi1) (Figure 4). Since Rho is known as a rate limiting protease of EGFR signaling and once EGFR signaling is activated, rho expression is further induced due to positive feedback regulation [26], Sarcoglycan complexes may regulate an upstream factor for Rhomboid. We also focused on the intriguing finding that dScgβ and dScgδ retain EGFlike consensus sequences in their C-terminus regions, and these sequences are well conserved between Drosophila and mammals (Figure 1B). Therefore, dScgβ and dScgδ might directly interact with doi: 10.7243/2050-0874-1-7 EGFR via the EGF-like consensus sequences and thus negatively regulate EGFR signaling. In order to assess this hypothesis, we investigated binding between Drosophila EGFR (CG10079) and dScgβ or dScgδ in vitro (Figure 6). The soluble version of the human EGFR extracellular domain, comprising amino acid residues 1-501, has been reported to show strong binding affinity with well-known EGFR ligands such as human EGF or TGFα [27]. Therefore, for in vitro binding assays, we generated a truncated and soluble form of Drosophila EGFR (Egfr1-559) composed of amino acid residues 1-559, which correspond to the human EGFR extracellular domain (Figure 6A). On Western blot analysis, Egfr1-559 appeared at 63kDa, consistent with the estimated molecular weight 62.9kDa (Figure 6B). While no band was detectable by anti-Flag antibody in the elution sample of the binding assay between dScgα and Egfr1-559 (Figure 6B-I, lane xii), Flag tagged Egfr1-559 bands were detected as coprecipitated products with His tagged full lengths of dScgβ or dScgδ (Figure 6B-II and IV, lane xii). 5.3% of Flag-dScgβ and 22.3% of Flag-dScgδ in the Input extracts were eluted. For further analysis, we tested the binding between Egfr1-559 and truncated forms of dScgβ or dScgδ lacking EGF-like consensus sequences, namely dScgβΔEGF-like and dScgδΔEGF-like. Neither was detectable in the coprecipitated fraction with Egfr1-559 (Figure 6B-III and V, lane xii), indicating that dScgβ and dScgδ directly associate with the EGFR extracellular domain via their EGF-like consensus sequences. Taking into account the finding that knockdown of all three sarcoglycans in each case caused alteration in rhomboid expression in pupal retinae (Figure 4), we conclude that Sarcoglycans work as a whole complex in negative regulation of EGFR signaling through direct association of the EGF consensus sequences in dScgβ and dScgδ with the extracellular domain of EGFR in Drosophila. Argos and spitz expression in the pupal retinae Both Spitz and Argos are known as regulatory proteins of EGFR signaling in Drosophila. While Spitz is a positively acting ligand, Argos acts in negative regulation by interrupting the association of Spitz with EGFR [27]. Intriguingly, we detected both Argos and Spitz in the two extra cells per each ommatidium, which apparently showed ectopic expression of rhomboid in dscgβ knockdown flies [19] (Figure 4), both in the control flies and in the knockdown flies (Figure 7). Notably for Spitz, expression was detected only in these two cells but not in photoreceptor cells. Knockdown of Sarcoglycans may therefore trigger secretion of an activated form of Spitz in these two cells presumably as a consequence of primary defects in the proper inactivation of EGFR. This could abnormally accelerate over-activation of EGFR signaling by positive-feedback mechanism. Discussion In recent years, there are increasing number of the reports suggesting the cross talk between DGC and EGFR. For instance, genetic modifier screening using the visible wing vein phenotype of dystrophin and dystroglycan RNAi flies identified two genes, argos and kekkon-1 which both encode negative regulators of EGFR signaling [27-30]. 9 Yamaguchi et al. HOAJ Biology 2012, http://www.hoajonline.com/journals/pdf/2050-0874-1-7.pdf doi: 10.7243/2050-0874-1-7 Both dystrophin and dystroglycan mutants suppressed the posterior as reduction of Dystroglycan levels in normal cells does not induce crossvein defective phenotype [30]. Moreover, EGFR signaling has tumor formation [39,41] and none of the DGC related muscular been reported to regulate expression of dystroglycan in Drosophila dystrophies is reported to be linked with remarkable increase in tumor oocytes [31]. We have also proposed in a previous report that dScgβ occurrence in human patients. However, Dystroglycan at least appears interacts with EGFR signaling in Drosophila [19]. The data suggested to act as a tumor suppressor since re-expression of dystroglycan in a close relationship between DGC and EGFR signaling in a variety of breast cancer cells, in which dystroglycan expression was originally tissues. However, the molecular mechanisms have remained elusive. decreased, significantly reduced tumor size [42]. Furthermore, it Our present in vitro binding analysis of Drosophila Sarcoglycans and is well known that EGFR signaling is over-activated and/or missEGFR provides evidence of the linking mechanism. regulated in many solid tumors [43]. Abnormal activation of EGFR EGFR signaling needs to be controlled precisely throughout signaling triggers processes that promote tumor cell proliferation, animal development [32]. Especially, it is well known that the control migration, and angiogenesis. It is of interest that, also with respect of the proper level of EGFR signaling is vital for eye development in to tumor genesis, the DGC and EGFR signaling present reciprocal Drosophila since either hyperactive or hypoactive EGFR signaling actions. Further elucidation of interplay between DGC and EGFR causes abnormalities [33]. Hence, in order to adjust levels strictly, signaling during tumor progression might shed light on novel EGFR signaling receives complex modulation by multiple regulatory strategies for better management of cancer therapy by exploiting mechanisms, which must be coordinated [33,34]. In fact, there are DGC activity against EGFR signaling [43]. a variety of ligands with distinct properties and interactions exist between EGFR signaling and other signaling pathways, such as Conclusions the Notch pathway, Insulin receptor/Target of rapamycin kinase In vitro data in combination with immunohistochemical data for pathways, and the Frizzled/PCR pathway [35-37]. Sarcoglycans sarcoglycan knockdown fly retinae, we propose that Sarcoglycan dependent negative regulation is also likely from the data presented complex formation is required for eye development. Notably, the in this report. Sarcoglycans appear to work for inactivation of EGFR direct binding of β-Sarcoglycan and δ-Sarcoglycan to EGFR might via direct association in pupal retinae (Figures 4 and 6). dScgβ be critical for the proper regulation of EGFR signaling during eye and dScgδ, both of which retain EGF-like consensus sequences development in Drosophila. in their C-termini, showed binding to EGF binding region of EGFR (Figure 6B-II and IV). In contrast, dScgα, which carries no EGF- Competing interests like consensus sequence, did not display binding (Figure 6B-II). The authors declare that they have no competing interests. Furthermore, the truncated forms of dScgβ and dScgδ lacking Authors’s contribution RH participated in designing experiments, carried on in the EGF-like consensus lost the binding ability (Figure 6B-III and vitro binding assays and immunostaining experiments. V). These EGF-like consensus sequence in dScgβ and dScgδ may MY carried out establishment of transgenic flies. HY competitively inhibit Spitz binding to EGFR. Therefore, we suggest carried out genetic interaction analyses. RH, HY and that the negative regulation of EGFR signaling is, at least in part, MY participated in writing the manuscript. Authors’s information controlled by direct association of EGFR with dScgβ and dScgδ. It has been reported that ectopic expression of dystroglycan is HY is assistant professor and full professor at the 1Department of Applied Biology and induced in both egfr mutant clone cells and in null mutant clone MY 3 Insect Biomedical Research Center, Kyoto Institute of Technology. cells of Ras in posterior follicle cells, whereas, mis-expression of RH was a post-doc at 2Venture laboratory, the constitutively active form of EGFR caused down-regulation of Kyoto Institute of Technology. dystroglycan expression in anterior follicle cells. Both observations Acknowledgements are consistent in suggesting that EGFR signaling regulates expression The authors are grateful to Dr. Y Hiromi for kindly providing of dystroglycan during oogenesis [31]. Dystroglycan plays a role in fly stocks, Dr. S Taketani for advice in using pETDuet-1 system, determining cellular polarity and in axis formation during oogenesis Mr K. Ogata and Mr T. Ito for technical assistance and Dr. M. Moore for comments on the English manuscript. This study [31,38]. An adequate level of Dystroglycan expression controlled was supported in part by a Venture Laboratory grant in Kyoto by EGFR signaling and a gradient in Dystroglycan expression could Institute of Technology and Grants-in-Aid from the Ministry of be required to establish the anterior-posterior axis in oogenesis Education, Culture, Sports, Science and Technology of Japan. [31]. Feedback regulation from the DGC side to cause up-regulated The Elav monoclonal antibody developed by Rubin, G.M. EGFR signaling might contribute to maintaining this gradient and the Lamin monoclonal antibody contributed by Fisher, P.A. were kindly supplied from the Developmental Studies of Dystroglycan expression. In the eyes, the breakdown of the Hybridoma Bank developed under the auspices of the NICHD DGC induced by knockdown of sarcoglycans might stimulate the and maintained by The University of Iowa, Department of feedback effect and lead to over-activation of EGFR signaling as Biology, Iowa City, IA 52242. We also acknowledge Bloomington stock center for supplies of the mutant fly lines, and Berkeley observed in this study. Interestingly, several published studies have shown that no or Drosophila Genome Project for the cDNA containing EST clones. reduced expression of α-dystroglycan or β-dystroglycan frequently Publication history occurs in human cancers [38-40]. Alteration of dystroglycan Received: 21-May-2012 Revised: 13-June-2012 expression is unlikely to be the primary reason for tumorigenesis Accepted: 09-July-2012 Published: 27-July-2012 10 Yamaguchi et al. HOAJ Biology 2012, http://www.hoajonline.com/journals/pdf/2050-0874-1-7.pdf doi: 10.7243/2050-0874-1-7 References 1. 2. Lapidos KA, Kakkar R, McNally EM: The dystrophin glycoprotein complex: signaling strength and integrity for the sarcolemma. Circ Res 2004; 94;(8.);1023-31. | Aricle | PubMed Roberds SL, Leturcq F, Allamand V, Piccolo F, Jeanpierre M, Anderson RD, et al.: Missense mutaions in the adhalin gene linked to autosomal recessive muscular dystrophy. Cell 1994; 78;(4.);625-3. | Aricle | PubMed Curr Opin Genet Dev 2002; 12;(3.);349-61. | Aricle | PubMed 18. Allikian MJ, Hack AA, Mewborn S, Mayer U, McNally EM: Geneic compensaion for sarcoglycan loss by integrin alpha7beta1 in muscle. J Cell Sci 2004; 117;(Pt 17.);3821-30. | Aricle | PubMed 19. Hashimoto R, Yamaguchi M: Geneic link between beta-sarcoglycan and the Egfr signaling pathway. Biochem Biophys Res Commun 2006; 348;(1.);212-21. | Aricle | PubMed 3. Lim LE, Duclos F, Broux O, Bourg N, Sunada Y, Allamand V, et al.: Betasarcoglycan: characterizaion and role in limb-girdle muscular dystrophy linked to 4q12. Nat Genet 1995; 11;(3.);257-65. | Aricle | PubMed 20. Holt KH, Campbell KP: Assembly of the sarcoglycan complex. Insights for muscular dystrophy. J Biol Chem 1998; 273;(52.);34667-70. | Aricle | PubMed 4. Noguchi S, McNally EM, Ben Othmane K, Hagiwara Y, Mizuno Y, Yoshida M, et al.: Mutaions in the dystrophin-associated protein gammasarcoglycan in chromosome 13 muscular dystrophy. Science 1995; 270;(5237.);819-22. | Aricle | PubMed 21. Shi W, Chen Z, Schotenfeld J, Stahl RC, Kunkel LM, Chan YM: Speciic assembly pathway of sarcoglycans is dependent on beta- and deltasarcoglycan. Muscle Nerve 2004; 29;(3.);409-19. | Aricle | PubMed 5. Nigro V, Moreira EdS, Piluso G, Vainzof M, Belsito A, Politano L, et al.: Autosomal recessive limbgirdle muscular dystrophy, LGMD2F, is caused by a mutaion in the [delta]-sarcoglycan gene. Nat Genet 1996; 14;(2.);195-8. | Aricle 6. Einger AJ, Feng G, Sanes JR: epsilon-Sarcoglycan, a broadly expressed homologue of the gene mutated in limb-girdle muscular dystrophy 2D. J Biol Chem 1997; 272;(51.);32534-8. | Aricle | PubMed 7. Wheeler MT, Zarnegar S, McNally EM: Zeta-sarcoglycan, a novel component of the sarcoglycan complex, is reduced in muscular dystrophy. Hum Mol Genet 2002; 11;(18.);2147-54. | Aricle | PubMed 24. Robertson HM, Preston CR, Phillis RW, Johnson-Schlitz DM, Benz WK, Engels WR: A stable genomic source of P element transposase in Drosophila melanogaster. Geneics 1988; 118;(3.);461-70.. | Aricle | PubMed Abstract | PubMed Full Text 8. Vainzof M, Passos-Bueno MR, Canovas M, Moreira ES, Pavanello RC, Marie SK, et al.: The sarcoglycan complex in the six autosomal recessive limb-girdle muscular dystrophies. Hum Mol Genet 1996; 5;(12.);19639. | Aricle | PubMed 25. Hashimoto R, Yamaguchi M: Dynamic changes in the subcellular localizaion of Drosophila beta-sarcoglycan during the cell cycle. Cell Struct Funct 2006; 31;(2.);173-80. | Aricle | PubMed 9. Cohn RD, Campbell KP: Molecular basis of muscular dystrophies. Muscle Nerve 2000; 23;(10.);1456-71. | Aricle | PubMed 22. Takahashi Y, Hirose F, Matsukage A, Yamaguchi M: Ideniicaion of three conserved regions in the DREF transcripion factors from Drosophila melanogaster and Drosophila virilis. Nucleic Acids Res 1999; 27;(2.);510-6. | Aricle | PubMed Abstract | PubMed Full Text 23. Spradling AC, Rubin GM: Transposiion of cloned P elements into Drosophila germ line chromosomes. Science 1982; 218;(4570.);341-7. | Aricle | PubMed 26. Freeman M: Feedback control of intercellular signalling in development. Nature 2000; 408;(6810.);313-9. | Aricle | PubMed 10. Greener MJ, Roberts RG: Conservaion of components of the dystrophin complex in Drosophila. FEBS Let 2000; 482;(1-2.);13-8. | Aricle | PubMed 27. Garret TP, McKern NM, Lou M, Elleman TC, Adams TE, Lovrecz GO, et al.: Crystal structure of a truncated epidermal growth factor receptor extracellular domain bound to transforming growth factor alpha. Cell 2002; 110;(6.);763-73. | Aricle | PubMed 11. Shiga K, Yoshioka H, Matsumiya T, Kimura I, Takeda S, Imamura M: Zeta-sarcoglycan is a funcional homologue of gamma-sarcoglycan in the formaion of the sarcoglycan complex. Exp Cell Res 2006; 312;(11.);2083-92. | Aricle | PubMed 28. Ghiglione C, Carraway KL, 3rd, Amundadoir LT, Boswell RE, Perrimon N, Dufy JB: The transmembrane molecule kekkon 1 acts in a feedback loop to negaively regulate the acivity of the Drosophila EGF receptor during oogenesis. Cell 1999; 96;(6.);847-56. | Aricle | PubMed 12. Allikian MJ, Bhabha G, Dospoy P, Heydemann A, Ryder P, Earley JU, et al.: Reduced life span with heart and muscle dysfuncion in Drosophila sarcoglycan mutants. Hum Mol Genet 2007; 16;(23.);2933-43. | Aricle | PubMed 29. Ghiglione C, Amundadoir L, Andresdoir M, Bilder D, Diamoni JA, Noselli S, et al.: Mechanism of inhibiion of the Drosophila and mammalian EGF receptors by the transmembrane protein Kekkon 1. Development 2003; 130;(18.);4483-93. | Aricle | PubMed 13. Chen J, Shi W, Zhang Y, Sokol R, Cai H, Lun M, et al.: Ideniicaion of funcional domains in sarcoglycans essenial for their interacion and plasma membrane targeing. Exp Cell Res 2006; 312;(9.);1610-25. | Aricle | PubMed 30. Kucherenko MM, Pantoja M, Yatsenko AS, Shcherbata HR, Fischer KA, Maksymiv DV, et al.: Geneic modiier screens reveal new components that interact with the Drosophila dystroglycan-dystrophin complex. PLoS One 2008; 3;(6.);e2418. | Aricle | PubMed Abstract | PubMed Full Text 14. Crosbie RH, Lebakken CS, Holt KH, Venzke DP, Straub V, Lee JC, et al.: Membrane targeing and stabilizaion of sarcospan is mediated by the sarcoglycan subcomplex. J Cell Biol 1999; 145;(1.);153-65. | Aricle | PubMed Abstract | PubMed Full Text 15. Durbeej M, Cohn RD, Hrstka RF, Moore SA, Allamand V, Davidson BL, et al.: Disrupion of the beta-sarcoglycan gene reveals pathogeneic complexity of limb-girdle muscular dystrophy type 2E. Mol Cell 2000; 5;(1.);141-51. | Aricle | PubMed 16. Straub V, Duclos F, Venzke DP, Lee JC, Cutshall S, Leveille CJ, et al.: Molecular pathogenesis of muscle degeneraion in the delta-sarcoglycan-deicient hamster. Am J Pathol 1998; 153;(5.);1623-30. | Aricle | PubMed Abstract | PubMed Full Text 17. Durbeej M, Campbell KP: Muscular dystrophies involving the dystrophin-glycoprotein complex: an overview of current mouse models. 31. Poulton JS, Deng WM: Dystroglycan down-regulaion links EGF receptor signaling and anterior-posterior polarity formaion in the Drosophila oocyte. Proc Natl Acad Sci U S A 2006; 103;(34.);12775-80. | Aricle | PubMed Abstract | PubMed Full Text 32. Shilo BZ: Regulaing the dynamics of EGF receptor signaling in space and ime. Development 2005; 132;(18.);4017-27. | Aricle | PubMed 33. Freeman M: Complexity of EGF receptor signalling revealed in Drosophila. Curr Opin Genet Dev 1998; 8;(4.);407-11. | Aricle | PubMed 34. Shilo BZ: Signaling by the Drosophila epidermal growth factor receptor pathway during development. Exp Cell Res 2003; 284;(1.);140-9. | Aricle | PubMed 35. Doroquez DB, Rebay I: Signal integraion during development: mecha- 11 Yamaguchi et al. HOAJ Biology 2012, http://www.hoajonline.com/journals/pdf/2050-0874-1-7.pdf doi: 10.7243/2050-0874-1-7 nisms of EGFR and Notch pathway funcion and cross-talk. Crit Rev Biochem Mol Biol 2006; 41;(6.);339-85. | Aricle | PubMed 36. McNeill H, Craig GM, Bateman JM: Regulaion of neurogenesis and epidermal growth factor receptor signaling by the insulin receptor/target of rapamycin pathway in Drosophila. Geneics 2008; 179;(2.);843-53. | Aricle | PubMed Abstract | PubMed Full Text 37. Weber U, Pataki C, Mihaly J, Mlodzik M: Combinatorial signaling by the Frizzled/PCP and Egfr pathways during planar cell polarity establishment in the Drosophila eye. Dev Biol 2008; 316;(1.);110-23. | Aricle | PubMed Abstract | PubMed Full Text 38. Brennan PA, Jing J, Ethunandan M, Gorecki D: Dystroglycan complex in cancer. Eur J Surg Oncol 2004; 30;(6.);589-92. | Aricle | PubMed 39. Sgambato A, Brancaccio A: The dystroglycan complex: from biology to cancer. J Cell Physiol 2005; 205;(2.);163-9. | Aricle | PubMed 40. Cross SS, Lippit J, Mitchell A, Hollingsbury F, Balasubramanian SP, Reed MW, et al.: Expression of beta-dystroglycan is reduced or absent in many human carcinomas. Histopathology 2008; 53;(5.);561-6. | Aricle | PubMed 41. Sgambato A, Migaldi M, Montanari M, Camerini A, Brancaccio A, Rossi G, et al.: Dystroglycan expression is frequently reduced in human breast and colon cancers and is associated with tumor progression. Am J Pathol 2003; 162;(3.);849-60. | Aricle | PubMed Abstract | PubMed Full Text 42. Muschler J, Levy D, Boudreau R, Henry M, Campbell K, Bissell MJ: A role for dystroglycan in epithelial polarizaion: loss of funcion in breast tumor cells. Cancer Res 2002; 62;(23.);7102-9. | Aricle | PubMed 43. Laskin JJ, Sandler AB: Epidermal growth factor receptor: a promising target in solid tumours. Cancer Treat Rev 2004; 30;(1.);1-17. | Aricle | PubMed Citation: Hashimoto R, Yoshida H and Yamaguchi M: Sarcoglycan complex formation is involved in regulation of EGFR signaling during Drosophila eye development. HOAJ Biology 2012, 1:7. http://dx.doi.org/10.7243/2050-0874-1-7 12