Basic HTML Version
Full Abstracts – CELL BIOLOGY AND SIGNAL TRASDUCTION I
156
98
Essential and equivalent roles for ligands signaling through the EGFR in Drosophila development.
Christina L. Austin, Amanda A. Simcox.
Department of Molecular Genetics, The Ohio State University, Columbus, OH.
Drosophila
Epidermal Growth Factor Receptor (dEGFR), the sole fly homolog of the vertebrate EGFR/ErbB receptor family, is a receptor tyrosine kinase
that signals through the canonical Ras/MAPK pathway, mediating diverse developmental processes by communicating both proliferative and differentiation
cues. Dysfunction of the pathway is commonly implicated in human cancers, including amplification of the orphan receptor ErbB2/Neu. dEGFR shares a
structural configuration with ErbB2/Neu, which differs from that of other ErbBs, and has been described as ‘poised’ to dimerize in the absence of ligand
binding. Here we provide definitive evidence that dEgfr is absolutely dependent on its ligands for signaling: quadruple mutant embryos that lack all EGF-
like ligands are phenotypically indistinguishable from receptor null embryos, ruling out the possibility that an unliganded fly receptor is active. This
comparison reveals a previously undescribed role for the ligand
keren (krn)
in early development, and further investigation reveals a cryptic role for krn in
wing development. Yet, while essential, the ligands do not appear to have unique roles. Surprisingly, we found that mutants of the neuregulin-like ligand
vein (vn)
can be rescued by expression of any one of the three TGFα-like ligands, if expressed under the control of the native
vn
promoter. Optimal rescue
was achieved with the higher-affinity TGFα-like ligands by reducing the dose. This functional equivalence suggests, therefore, that unique outcomes
attributed to each ligand are more dependent on expression pattern (and cellular context) than qualitatively distinct properties of individual ligands and
favors the idea that different signaling outcomes are purely quantitative effects.
99
Trafficking of the EGFR ligand Spitz to distinct membrane domains regulates signaling capacity in polarized tissues.
Josefa Steinhauer
1
, Jessica
Treisman
2
. 1) Department of Biology, Yeshiva College, New York, NY; 2) Skirball Institute of Biomolecular Medicine, NYU Langone Medical Center,
New York, NY.
EGFR ligands undergo complex processing during their maturation to active signaling proteins. Like the mammalian ligands, the predominant
Drosophila
EGFR ligand Spitz (Spi) is produced as a transmembrane precursor. In the secretory pathway, Spi is proteolyzed within its transmembrane domain, and the
soluble active ligand is released into the secretory lumen. The soluble ligand has been shown to be palmitoylated in a late secretory compartment, and
palmitoylation mediates membrane association at the cell surface following secretion. We have found that the precursor can reach the cell surface in the
absence of proteolysis but is unable to signal, even when it is not palmitoylated. It is unclear why the transmembrane precursor is inactive, while membrane
association of the processed ligand via the palmitate group promotes activity. We generated a panel of chimeric Spi constructs containing the Spi
extracellular region and exogenous transmembrane domains. All the chimeras can activate the EGFR, despite varying orientation and distance of the Spi
domain from the membrane. Thus, the presence of a transmembrane domain does not inhibit signaling, and orientation of the Spi domain with respect to the
membrane is not determinative. Despite the fact that the Spi transmembrane precursor is unable to activate the EGFR in polarized imaginal tissues, we have
found that it can signal in a tissue culture assay. Thus, tissue polarity may be a determinant for signaling ability in vivo. In support of this hypothesis, we
have found that our chimeric Spi constructs localize to the basolateral membrane, whereas unprocessed Spi precursor localizes apically. We currently are
investigating whether active processed Spi and the EGFR also localize basolaterally, and whether the intracellular domain of the precursor contains apical
targeting sequences. Together, our data support the model that productive signaling is limited to the basolateral compartment.
100
Mechanisms of Evi/Wls mediated Wnt-secretion - novel pathways beyond bulk secretion.
Julia C. Gross, Varun Chaudhary, Michael Boutros. Division
Signaling and Functional Genomics, German Cancer Research Center (DKFZ), Heidelberg, Germany.
During development, cells need to communicate to transform an undifferentiated sheet of cells into a functional, heterogenous type of tissue. This is
achieved by spatially restricted secretion of morphogens, such as proteins of the Wnt family. These secreted glycoproteins are required for a variety of
developmental processes, which are highly conserved from fly to human. How hydrophobic Wnt proteins spread in the extracellular space has been a long-
standing question. Different hypotheses have been proposed to explain how Wnt could travel in the extracellular space, such as coating of lipoprotein
particles and/or as part of secreted vesicles. Nevertheless, the mechanism of Wnt secretion and gradient formation remains poorly understood. Furthermore,
a complete set of proteins involved in various aspects of Wnt trafficking still remains to be identified. We have previously identified the transmembrane
protein Eveness interrupted (Evi/Wls) as a core component of the Wnt secretion process. Evi shuttles Wnt proteins from the Golgi to the plasma membrane
and is then recycled by the retromer complex. Newer data suggest that Evi/Wnt complexes are not just dissociating at the plasma membrane and that Evi, as
a multipass membrane protein can transfer from one cell to another. Our current research focuses on understanding how and when Evi releases Wnt on the
cellular level. We are interested in finding additional factors required for further processing of Wnt proteins and trafficking of Evi/Wnt complexes for the
secretion of functional signaling entities. Here we present our data on this novel functional role of Evi in Wnt secretion and our RNAi screening approaches
along with candidates that are under investigation.