The genetic synergy between Ed and Neuroglian (Nrg) suggests that both proteins might also physically interact with each other. Using antibodies that specifically recognize Ed and both isoforms of Nrg for an immunocytochemical analysis, this possibility was first tested by examining Nrg and Ed expression patterns in the developing Drosophila eye disc. Both colocalize to all cells throughout the third instar larval eye disc, including undifferentiated cells and developing ommatidial clusters (Chandra, 2003).
Similar to several vertebrate Ig-domain CAMs, which interact with members of the L1 family, Ed might exhibit both homophilic and heterophilic adhesive activities. Genetic results support this possibility. To investigate the adhesive function of Ed, Ed protein was expressed in Drosophila Schneider 2 (S2) cells. Owing to their lack of endogenous CAMs, S2 cells have been successfully used for the functional analysis of a range of adhesive proteins. HA epitope-tagged Ed protein is expressed by S2 cells at a high and stable level. This epitope-tagged version of the Ed protein exhibits an apparent molecular weight of about 160 kDa, about the size to be expected from the Ed amino acid sequence. When S2 cells expressing Ed protein are allowed to aggregate, they formed small to medium-sized cell clusters. In order to establish whether this cell aggregation is due to a homophilic activity of Ed or to a heterophilic interaction with an endogenous ligand, which might be expressed on the S2 cell surface, cell-mixing experiments were performed. Aggregation experiments, in which unlabeled, Ed-expressing cells were mixed with DiI-labeled, Ed-expressing cells, resulted in mixed cell clusters, consisting of labeled and unlabeled cells. By contrast, DiI-labeled, native S2 cells that do not express Ed protein are not recruited into Ed cell aggregates. These results demonstrate that Ed acts as a homophilic adhesion protein and exhibits no heterophilic affinity to any endogenous S2 cell protein (Islam, 2003).
Mixing experiments were performed to determine whether Nrg and Ed engage in a heterophilic interaction. In comparison with Ed-expressing S2 cells, S2 cells expressing Nrg exhibit a much stronger homophilic cell adhesion capability, resulting in very large S2 cell aggregates. The co-aggregation of Ed-expressing cells with Nrg-expressing S2 cells yielded mixed cell aggregates, consisting of cells from both cell populations. DiI-labeled, native S2 cells are not incorporated into Nrg-cell clusters and quantitative evaluation demonstrates that the heterophilic interaction of Ed is specific to S2 cells expressing Nrg and that Ed does not interact with other Drosophila adhesion molecules, such as Drosophila Fasciclin 1. Thus, Ed and Nrg engage in a robust and specific heterophilic trans-interaction (Islam, 2003).
The strength and stability of this interaction was demonstrated by co-immunoprecipitation experiments. Since native Nrg interacts with ankyrin and the S2 cell membrane skeleton, it becomes resistant to non-ionic detergent extraction after engaging in cell adhesion. Therefore, an artificial, GPI-anchored form of Nrg was used for these co-immunoprecipitation experiments. S2 cells that expressed Ed or NrgGPI were mixed, co-aggregated and subsequently extracted with Triton X-100. Soluble proteins and protein-complexes were immunoprecipitated with an anti-Nrg or a control antibody and analyzed on Western blots for the presence of Ed protein. Ed protein was efficiently co-immunoprecipitated together with NrgGPI, suggesting that a stable and tight interaction is formed between these two adhesive molecules (Islam, 2003).
The extracellular structure of immunoglobulin and fibronectin repeats in Echinoid suggests that it may be a member of the L1 class of cell-adhesion proteins, which are important in mediating axon guidance and cell fate decisions. To examine the ability of Echinoid to mediate homophilic adhesive interactions, cultured S2 cells were transfected with epitope-tagged versions of Echinoid. EdFLAG is capable of co-precipitating Edmyc, suggesting that Echinoid can form multimers. In the converse experiment, immunoprecipitation of Edmyc also co-precipitates EdFLAG. A form of EdFLAG lacking extracellular sequences (EdFLAGdeltaN) does not co-precipitate Edmyc, suggesting that the proteins associate through their extracellular domains. Binding of EdFLAG to Edmyc is unaffected by transfection with Egfr. These results are consistent with the potential of Echinoid to act as a cell adhesion molecule; in keeping with this, defects in R8 differentiation have been observed in wild-type cells just outside echinoid mutant patches of tissue (Rawlins, 2003). However, the nature of this potential adhesive interaction on signaling is not clear, since overexpression of Echinoid, which might be expected to affect adhesion, has no effect on Egfr activity (Spencer, 2003).
Another common property of L1 immunoglobulin proteins is cleavage by plasmin or ADAM-class proteases at sites within or near FN3 domains. A similar cleavage of Echinoid was observed in S2 cultured cells. Immunoprecipitations of C-terminally tagged Echinoid produces two bands on SDS-PAGE gels: one, which corresponds to full-length Echinoid, migrates as a 190 kDa protein (larger than the predicted molecular weight of 146 kDa, possibly owing to glycosylation); the other migrates at approximately 55 kDa, corresponding approximately to the C-terminal 500 amino acids of Echinoid. Consistent with this, an antibody to the N-terminal region of Echinoid detects a ~130 kDa form of the protein in media from transfected S2 cells. This suggests that, in addition to full-length Echinoid, cells also contain a truncated form which lacks most of the extracellular motifs, and that the Ig/Fibronectin domains of the protein are shed into the surrounding extracellular space. The cleavage appears to be constitutive in S2 cells, and is not altered by transfection of Egfr (Spencer, 2003).
Echinoid has been proposed to act in a pathway parallel to Egfr, with signaling converging at the nucleus (Bai, 2001). Data from the current study suggests, in contrast, that loss of echinoid acts upstream of the nucleus to stabilize ERKA phosphorylation. Given the localization of Echinoid at the cell surface (Bai, 2001) the potential for direct interactions between Echinoid and Egfr was examined in S2 cultured cells. FLAG-tagged Echinoid was immunoprecipitated and analyzed by Western blots: transfection with increasing amounts of Echinoid co-precipitates increasing amounts of Egfr, suggesting that this is a specific interaction. Similarly, Echinoid containing a Myc tag also co-precipitates Egfr. In the converse experiment, immunoprecipitation of Egfr co-precipitates EdFLAG. The site of binding between Echinoid and Egfr could not be shown conclusively from these experiments: both EdFLAGdeltaC and EdFLAGdeltaN efficiently immunoprecipitate Egfr, suggesting that their interaction occurs near the transmembrane domain or through multiple domains (Spencer, 2003).
L1 cell-adhesion proteins are frequently regulated by phosphorylation, which controls their binding to other cytoplasmic signaling proteins. The physical association of Echinoid with Egfr, a receptor tyrosine kinase, suggested that it might be a substrate for tyrosine phosphorylation. To examine this possibility, S2 cell culture was sued to assess tyrosine phosphorylation in response to Egfr signaling. Echinoid exhibits a low level of tyrosine phosphorylation in the absence of added Egfr. Co-transfection with Egfr, even without added ligand, leads to a dramatic increase in the phosphorylation of Echinoid. This phosphorylation is increased further by addition of media containing a soluble form of the activating Egfr ligand Spitz. Phosphorylation is limited to the tyrosine-rich intracellular region: a form of Echinoid lacking the intracellular domain (EdFLAGdeltaC) is not phosphorylated. Interestingly, EdFLAGdeltaN, which lacks the extracellular immunoglobulin and fibronectin motifs, is phosphorylated in response to Egfr signaling, suggesting that these extracellular motifs are unnecessary for phosphorylation to occur. Bai (2001) suggested that Echinoid acts in a pathway parallel to Egfr, and that their activities converge in the nucleus. This study finds, however, that expression of the activated Ras isoform dRas1Val12 does not lead to increased Echinoid phosphorylation, indicating that phosphorylation of Echinoid in response to Egfr signaling occurs upstream of Ras activation. These data do not resolve whether Echinoid is phosphorylated directly by Egfr or by cytoplasmic tyrosine kinases immediately downstream of its activity, an issue also unresolved for other cell-adhesion proteins. Phosphorylation of Echinoid in response to Egfr signaling was confirmed in vivo: Echinoid immunoprecipitated from w1118 eye discs (which are essentially wild type at this stage) shows a low level of phosphorylation. Echinoid immunoprecipitated from discs in which Rhomboid (an activator of Egfr) was transiently expressed shows an approximately fourfold increase in tyrosine phosphorylation. Transient expression of Argos, an inhibitor of Egfr, produces either no change or a slight decrease in Echinoid phosphorylation levels over wild type. The importance of this phosphorylation to the function of Echinoid is not clear from these data. It has been noted, however, that strong overexpression of Echinoid in the developing retina leads to a rough eye in adults (Bai, 2001). This phenotype requires the presence of the Echinoid intracellular domain (Bai, 2001), suggesting that this domain may be essential for the function of Echinoid (Spencer, 2003).
The cytokine-activated Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway plays an important role in the control of a wide variety of biological processes. When misregulated, JAK/STAT signaling is associated with various human diseases, such as immune disorders and tumorigenesis. To gain insights into the mechanisms by which JAK/STAT signaling participates in these diverse biological responses, a genome-wide RNA interference (RNAi) screen was carried out in cultured Drosophila cells. One hundred and twenty-one genes were identified whose double-stranded RNA (dsRNA)-mediated knockdowns affected STAT92E activity. Of the 29 positive regulators, 13 are required for the tyrosine phosphorylation of STAT92E. Furthermore, it was found that the Drosophila homologs of RanBP3 and RanBP10 are negative regulators of JAK/STAT signaling through their control of nucleocytoplasmic transport of STAT92E. In addition, a key negative regulator of Drosophila JAK/STAT signaling was identified, protein tyrosine phosphatase PTP61F; it is a transcriptional target of JAK/STAT signaling, thus revealing a novel negative feedback loop. This study has uncovered many uncharacterized genes required for different steps of the JAK/STAT signaling pathway (Baeg, 2005).
echinoid (ed) was identified as a positive regulator required for Upd-dependent STAT92E tyrosine phosphorylation. ed encodes a cell adhesion molecule and has been shown to be a negative regulator of the EGFR signaling pathway during Drosophila eye development. Previous experiments have shown both positive and negative interactions between the JAK/STAT pathway and the EGFR pathway. For example, STAT92E mutants phenocopy mutants in the EGFR pathway. Furthermore, studies using mammalian tissue culture systems have demonstrated that EGFR signaling activates both JAK1 and STAT1. In addition, EGFR-induced cell migration is mediated predominantly by the JAK/STAT pathway in primary esophageal keratinocytes. Similarly, ed has been shown to be responsible for defective cell migration in Caenorhabditis elegans. Therefore studying the role of ed in JAK/STAT signaling in different contexts may facilitate understanding of the genetic and biochemical mode of STAT activation by EGFR signaling, and provide insights into the mechanisms governing cancer cell metastasis in humans (Baeg, 2005).
Echinoid is an immunoglobulin domain-containing transmembrane protein that
modulates cell-cell signaling by Notch and the EGF receptors. In
the Drosophila wing disc epithelium, Echinoid is a component of adherens
junctions that cooperates with DE-Cadherin in cell adhesion. Echinoid and
β-catenin (a DE-Cadherin interacting protein) each possess a C-terminal
PDZ domain binding motif that binds to Bazooka/PAR-3; these motifs redundantly
position Bazooka to adherens junctions. Echinoid also links to actin filaments
by binding to Canoe/AF-6/afadin. Moreover, interfaces between
Echinoid- and Echinoid+ cells, like those between
DE-Cadherin- and DE-Cadherin+ cells, are deficient
in adherens junctions and form actin cables. These characteristics probably
facilitate the strong sorting behavior of cells that lack either of these
cell-adhesion molecules. Finally, cells lacking either Echinoid or DE-Cadherin
accumulate a high density of the reciprocal protein, further suggesting that
Echinoid and DE-Cadherin play similar and complementary roles in cell adhesion (Wei, 2005).
Several observations prompted the study of Ed as a canonical CAM in the
monolayered wing imaginal disc. Thus, mitotic recombination clones of cells
mutant for the null allele ed1x5 exhibit rounded and smooth
contours, in contrast to clones of wild-type
cells that show wiggly shapes. This indicated that
ed- /- cells have distinct
adhesive properties and assort with themselves rather than with the surrounding
ed+/- M+/- cells.
(ed1x5 clones were M+,
since without a growth advantage they hardly survive). It was also observed that Ed was absent
from the membrane of the heterozygous cells that contacted the mutant cells,
a finding consistent with the
observation that Ed forms homophilic interactions and that these are required to
incorporate/stabilize Ed at the cell membrane. Finally, Ed was found to
localize basally to the apical marker Crb and apically to the basolateral marker Dlg.
In fact, Ed colocalizes with both DE-Cad and Arm,
and, therefore, it might be part of AJs. AJs are structures important for
cell-cell contact and recognition. So, these results suggested that Ed plays a
role in cell-cell adhesion (Wei, 2005).
Whether Ed affects components of AJs was examined by analyzing the localization of Arm within ed mutant clones. Arm strongly accumulates at the apical membranes of ed- /- cells, and these cells
have a reduced apical surface.
Both effects are clear in small clones, but cells within larger clones (over hundreds of cells) had both
the density of Arm and the apical surface more similar to those of the wild-type
cells. Similar observations were made with DE-Cad and Actin. It is suggested that the increased concentration of these molecules in small clones most probably results from the apical constriction as supported by the accumulation of nonmuscle myosin II, without a net per cell increment of these proteins.
Alternatively, it could result from increased stability of these proteins. The
apical constriction continued through the SJs and ended at the planes just below
the GJs as revealed by an Innexin antibody. Hence, these
ed- /- cells adopt a bottle
shape. In contrast, the apposed ed- /- and
ed+/- cells that form the border of the clone enlarge
and adopte a rectangular shape. At this interface, the
ed- /- cells often contacted the
heterozygous cells by their long sides, as if in an attempt to minimize the number
of cells that formed the interface (Wei, 2005).
Interestingly, Arm and DE-Cad, but not Actin,
are depleted at the interface membrane of both small and large clones. This suggests that ed- /- and ed
heterozygous cells discriminate one another and that AJs do not form properly in
between them (Wei, 2005).
ed clones are surrounded by an Actin 'cable'. High-magnification images
suggest that the cable is contained within the ed heterozygous cells
surrounding the clone and that it is therefore generated by these cells. Several observations
suggest that this Actin cable exerts a force. The cells surrounding an ed
clone elongate toward the clone and accumulate nonmuscle myosin II at the
interface membrane, as if attempting to cover the space exposed by the apically constricted
ed- /- cells. This effect is
reminiscent of the stretching of the leading-edge cells that will cover the
underlying amnioserosa during dorsal closure of the embryos. In the wing disc,
the boundary that separates the dorsal (D) and ventral (V) regions of the wing
pouch has the shape of a smooth arc and contains an actin 'fence'.
After the second instar, this boundary corresponds to a compartment border that
imposes absolute restrictions to cell lineages. Large
ed- /- clones close to or
touching this boundary displace it toward the clones. In contrast, ed clones that
straddle the boundary do not overtly distorted it, although the boundary could be less
smooth within the clone. (Straddling clones
might be originated before the compartment border was established or might be
formed of D and V clones that fuse together). Moreover, the Actin
cable surrounding the clones fuse with the Actin fence at the D/V
boundary, suggesting that the distortion of this boundary is effected through
this Actin linkage. Control
ed+ M+
clones do not induce such distortions. These observations
suggest that the Actin cable may contribute to the roundish shape of the
ed clones and help confine their cells (Wei, 2005).
DE-Cad is a classical homophilic cell adhesion molecule of
AJs. It interacts with β-catenin/Arm, which in turn binds α-catenin.
Through the association between α-catenin and F-Actin, DE-Cad establishes
links between cells that connect to the Actin cytoskeleton. This study shows that Ed
is another CAM that, at the resolution of confocal microscopy, is
also located at the AJs of imaginal disc cells. While cells in clones mutant for
ed still seem to form normal AJs, the cells at the border of the clone
seem impaired in forming them. It is hypothesized that this may help them segregate
from surrounding ed+/- cells. Ed was identified as a
binding partner for PDZ proteins that, similarly to Arm, helps localize Baz to
AJs. Moreover, it was found that through the binding of Cno, Ed, like
DE-Cad/β-catenin, may link to F-Actin. Hence, Ed has functions in
cell-cell adhesion similar to those of DE-Cad (Wei, 2005).
The differential adhesion hypothesis proposes that cell sorting may be driven by differences in
the quantity and/or quality of adhesive molecules displayed on the surface of
cells. In keeping with this hypothesis, it was found that
ed- /- cells sort out from
ed+/- cells, as shown by the remarkably round shapes
and smooth contours of the ed clones. Moreover, their differential
adhesiveness is also manifest by the fusion of different ed clones to
yield composite but still roundish clones. It is suggested that contraction of the
apically enriched Actin network and of the actin cable surrounding the clone,
possibly by interaction with nonmuscle myosin II also present there, may
contribute to the the apical constriction of the
ed- /- cells. It was also observed
that the interface between ed+/- and
ed- /- cells is depleted of
DE-Cad, Arm and Baz, besides completely lacking Ed. This strongly suggests that
this interface is deficient in AJs and probably helps to insulate
ed- /- cells from the
surrounding ed heterozygous cells. It is hypothesized that this deficiency of
AJs, which may reduce adhesion between ed+/- and
ed- /- cells, and
the inward-pulling force generated by apical constriction and the actin cable
may help create the smooth and rounded contour of the clones at the level of
AJs. At the plane of SJs, the clonal boundary is not as smooth. This may be due
to the presence of normal levels of SJs, since seemingly wild-type amounts of
Dlg were detected at the interface membrane. Normal levels of SJs may allow the
clones to remain integrated in the epithelium. It is stressed that when ed
clones grow large, the apical constriction disappears, suggesting that the
forces responsible for this constriction become insufficient or no longer
operate. If the force is exerted, at least in part, by the Actin cable
surrounding the clone, as in a purse-string mechanism, it would make sense that
this force becomes ineffectual as the number of cells within the clone
increases. Remarkably, these differences of apical cell constriction observed in
small and large ed clones have a correlate on the adult wing blade: small
clones display an increased density of trichomes, implying that their cells are
small or more tightly packed, whereas large clones have cells of normal size.
This indicates that the apical constriction is retained through imaginal disc
eversion, when the disc epithelium changes from columnar to planar (Wei, 2005).
In the embryonic
epithelium, Baz, localized to both AJs and the marginal zone, is the initial
apical regulator. How
is Baz recruited to the apical domain? In the follicular epithelium, Baz is
localized to this domain through lateral exclusion mediated by PAR-1/14-3-3 and
apical anchoring by Crb/Sdt/Patj. The data support an additional mechanism to localize
Baz to the apical domain. Both Ed and Arm can bind Baz through their C-terminal
PDZ binding motif and therefore they may redundantly localize Baz to AJs.
Indeed, the localization of Baz to AJs is relatively normal in the absence of
either one. Most Baz is lost only when both Arm and Ed are depleted, as
occurs at the interface membrane of ed clones or in large shg
clones where Ed gradually breaks down. In the latter case, there is good
colocalization between Baz and the sites maintaining residual Ed. It is suggested
that in the epithelium of the wing disc, Baz localizes to AJs by the combined
effects of its binding to Ed/Arm and the lateral exclusion of PAR-1/14-3-3.
Additionally, apical anchoring of Baz may be mediated by direct association
between the Baz and Crb apical complexes. During early embyogenesis, Ed is also
present at pseudocleavage furrows. This observation,
together with the ability of Ed to localize Baz to AJs, may explain the finding
that during cellularization, Baz can accumulate apically in the absence of Arm.
Ed also binds to the PDZ domain of Cno and mediates its localization to AJs, where Cno
interacts with F-Actin either directly or indirectly through the association
with Polychaetoid/ZO-1. Interestingly, the evolutionally conserved EIIV domain
of Ed binds Baz and Cno in a mutually exclusive manner. Thus, the concentrations
of and differential affinities between Ed, Baz, and Cno should determine their
dynamic equilibrium at AJs (Wei, 2005).
Although Baz is critical to form AJs in the
blastoderm and in the follicular epithelium, removal of Baz
(or Par-6) from cells of the wing disc does not affect the
localization of DE-Cad or Ed to AJs. This is consistent with the report that in
imaginal discs, Baz does not affect the localization of DE-Cad and Dlg but is
required for the asymmetric localization of cell fate determinants. Together, these
results suggest that in wing discs, the Baz complex is not critical for the
formation of AJs, and that the effect of the loss of Ed on AJs
formation/maintenance is not due to Baz depletion (Wei, 2005).
Several similarities between the roles of DE-Cad and Ed in the
wing disc epithelium are worth noting. Both Ed and DE-Cad are CAMs that
establish homophilic interactions and localize to AJs. The absence of either Ed
or of DE-Cad in cells of small clones causes their apical constriction and
strong segregation from wild-type cells, giving rise to smooth round borders. In
both cases, the mutant cells are impaired in forming AJs with neighboring
wild-type or heterozygous cells and are surrounded by an Actin cable. Ed
interacts with Cno, and DE-Cad with Arm, and both Cno and Arm directly or
indirectly associate with F-Actin. Thus, Ed and DE-Cad represent two distinct
classes of CAMs, with widely different chemical compositions, that connect to
F-Actin, contribute to cell adhesion in the wing disc, and seem to have
partially overlapping functions (Wei, 2005).
In contrast, DE-Cad and Ed differ in
their ability to regulate the apical/basal cell polarity. Ed affects components
of AJs, but not those of the apical Crb and the basolateral Dlg complexes. In
contrast, DE-Cadherin is necessary for Crb localization, but similarly to Ed, it
is not required for Dlg localization. Furthermore, the maintenance of Ed at AJs
requires DE-Cad. In contrast, localization of DE-Cad to AJs is independent of
Ed. Interestingly, the DE-Cad/Arm complex is not essential for the formation of
the follicular epithelium, but upon removal of this complex, the integrity of the
epithelium is lost slowly over the period of several days. This suggests that
other molecules may be maintaining the epithelial structure. During stages
1 to 10 of oogenesis Ed is mainly expressed in the follicle cells, and these cells,
if mutant for ed, show at low frequency a multilayered structure with
disrupted expression of some polarity markers. Thus,
it will be of interest to elucidate whether, in this epithelium, Ed and
DE-Cad/Arm also play partially redundant roles in cell adhesion and apical/basal
polarity. While both Ed and DE-Cad contribute to cell adhesion and recognition,
it is unclear whether each molecule imparts specific recognition properties to
cells, so that the final cell-cell affinity results from the sum of distinct
affinities mediated by these different CAMs. More specifically, can an increased
level (density) of DE-Cad replace the absence of Ed? The results showing that
ed- /- cells, with either normal
levels (in large clones) or high density (in small clones) of DE-Cad, do not
intermix with wild-type cells suggests that the binding specificity provided by a
given CAM is not overruled by a higher level (density) of a different CAM.
Moreover, the cell sorting properties conferred by Ed cannot account for the
separation of cells at both sides of the A/P compartment boundary of the wing
disc because A and P cells do not intermingle within composite ed, smo
double mutant clones. (Similarly, DE-Cad is not responsible for the sorting out
of A and P cells. Hence, cell-cell adhesion in the wing disc appears to depend on
multiple CAMs (Ed, DE-Cad, etc.), each imparting specific cell recognition
properties. Although Ed and its C-terminal EIIV motif are conserved in
invertebrates, no
clear vertebrate homolog with 7 Ig domains and a PDZ domain binding motif has
been found. Nectin1-4 comprises a family of 3 Ig domain-containing CAM that have
several differentially spliced forms and localize to AJs. Most spliced
forms share a conserved C-terminal E/A-X-Y-V that binds the PDZ domain of
Afadin. Moreover, this motif also interacts with Par-3, the vertebrate homolog
of Baz. In spite of these similarities, overexpression of either nectin 1-α or
3-α does not rescue the remarkable clonal phenotype of ed (Wei, 2005).
Glutamate receptor interacting protein (GRIP) homologues, initially characterized in synaptic glutamate receptor trafficking, consist of seven PDZ domains (PDZDs), whose conserved arrangement is of unknown significance. The Drosophila GRIP homologue (DGrip) is needed for proper guidance of embryonic somatic muscles towards epidermal attachment sites, with both excessive and reduced DGrip activity producing specific phenotypes in separate muscle groups. These phenotypes were utilized to analyze the molecular architecture underlying DGrip signaling function in vivo. Surprisingly, removing PDZDs 1–3 (DGripΔ1–3) or deleting ligand binding in PDZDs 1 or 2 convert DGrip to excessive in vivo activity mediated by ligand binding to PDZD 7. Yeast two-hybrid screening identifies the cell adhesion protein the Echinoid (Ed) type II PDZD-interaction motif as binding PDZDs 1, 2 and 7 of DGrip. ed loss-of-function alleles exhibit muscle defects, enhance defects caused by reduced DGrip activity and suppress the dominant DGripΔ1–3 effect during embryonic muscle formation. It is proposed that Ed and DGrip form a signaling complex, where competition between N-terminal and the C-terminal PDZDs of DGrip for Ed binding controls signaling function (Swan, 2006).
This study used genetics to develop a mechanistic model concerning a well-defined function mediated by Drosophila Grip—embryonic muscle guidance. Functional requirements were not transmitted by single domains, but were found to be distributed over the whole length of this 7 PDZD protein in an unexpectedly complex manner. Binding ligands via PDZDs 1 and 2 repressed the activity of the protein, binding to PDZD 3 was involved in de-repression, and PDZ-ligand binding via PDZD 7-mediated DGrip function after its de-repression. Despite the fact that there was no critical dependence on PDZDs 4–5 or interdomains for function, it cannot be excluded that interactions over these domains play a subthreshold role. In fact, the DGripΔ1–3x7 construct showed some residual functionality in terms of muscle rescue. Thus, the whole protein might be used as an 'intelligent frame' designed to execute fine controls such as thresholding functions or coincidence detections. In fact, all attempts to provide DGrip activity or to repress DGrip activities with only partial fragments (DGripΔ4–7, DGripΔ1–5) failed, leading to the belief that DGrip is responsible for the organization of a macromolecular complex, of which the transmembrane protein Ed is part (Swan, 2006).
This analysis suggests that a critical number of PDZDs are utilized for DGrip function, with both negative and positive interactions occurring. Such dependence between PDZDs may be due to structural chaperoning. Alternatively, a fixed orientation might be required for high-affinity binding to its targets as found for tandem PDZDs 1 and 2 in PSD-95, with a complex of two PDZDs having higher binding affinity than either PDZD alone. Moreover, allosteric changes upon PDZD-ligand binding could change binding affinities of neighboring domains or via bridging interactions where one molecule contacts multiple sites on a PDZ protein to effect conformational change. Such mechanisms might be the substrate for integrating ligand binding and functional output over a large 'multivalent' PDZD protein (Swan, 2006).
Point mutations of PDZD 1 and PDZD 2 recapitulated the DGripΔ1–3 phenotype in the lateral transverse muscles (LTM) group of muscles, indicating that the repressive function of the PDZDs 1–3 region is not 'structural' (i.e. by covering other PDZDs on the protein). Instead, it is suggested that ligand interactions are communicated over the whole protein to steer equilibrium between two different functional modes of DGrip signaling (Swan, 2006).
Ed was identified as a novel DGrip interactor. Ed is cell adhesion protein with 7 Ig and 2 FNIII domains, described to have both adherence and signaling roles in Drosophila tissues. It is highly conserved among invertebrates and its closest vertebrate homologues are Nectins, which exhibit 3 Ig domains and end in the PDZ-binding motif E/A-X-Y-V. Functionally, both protein families are similar: although not functionally redundant with Ed, Nectins are present at mammalian adherens junctions (AJs) along with l-afadin and, like Ed, regulate Cadherin-based adherence at AJs. Several lines of evidence link Ed to DGrip:Ed interacted with DGrip in a yeast two-hybrid screen, dependent on the C-terminal EIIV motif, mediated via PDZDs 1, 2 or 7. Myc-tagged DGrip specifically interacts with a peptide representing the last 10 amino acids of the Ed protein, including the EIIV PDZ-binding motif. ed zygotic mutants have defects in the morphogenesis of embryonic muscles qualitatively similar to DGripΔ1–3 overexpression. The dgripex36 muscle phenotype in embryos is enhanced by heterozygosity for ed1x5. Here, LTMs (unaffected in pure dgripex36) are affected as well. dgripex36 mutant muscles (both VLMs and LTMs) are sensitive to Ed overexpression. These synthetic defects suggest that DGrip, while itself not essential for LTM morphogenesis, regulates Ed in this group of muscles. Homozygosity for hypomorphic edslH8 chromosome strongly reduced the severity of the phenotype evoked by pan-muscular expression of DGripΔ1–3, indicating that Ed acts downstream of activated DGrip (Swan, 2006).
Notably, the pattern of Ed-PDZD binding correlates with the DGrip-dependent LTM phenotype. Expression of DGrip missing PDZDs 1, 2 and 3 together, or ligand binding in PDZD 1 and PDZD 2 only, showed a strong dominant active phenotype. Mutation of PDZD 2 caused a dominant phenotype in LTMs. In a yeast-two hybrid test, Ed interacted strongly with PDZD 2 with and PDZD 7, and more weakly with PDZD 1 (Swan, 2006).
In imaginal discs, Ed binds two different PDZD proteins via its EIIV motif: Canoe, an F-actin interacting protein and PAR-3/Bazooka. This interaction is mutually exclusive, thereby influencing cell adhesion and the remodeling of subcortical actin at AJs. This study proposes a similar mechanism, in that both functional states of Ed are established via binding to the same protein (DGrip) at different sites. In this model, DGrip may assist in maintaining equilibrium between active and inactive signaling states of Ed, which in its inactive state binds to PDZDs 1 and 2, and in its active form to PDZD 7 of DGrip. This interaction appears tissue specific in nature, since DGrip mutants do not display the full spectrum of defects of ed mutants (such as neurogenic phenotypes) and since there are as yet unknown members of the DGrip–Ed complex, such as that member that binds to the 'de-repressing' PDZD 3 (Swan, 2006).
Both Ed loss of function and overexpression can produce similar phenotypes in muscles, which are strongly enhanced by the absence of DGrip. Ed is described as a homophilic cell adhesion molecule, and is maternally expressed in the epidermis, over which nascent muscles 'crawl' during the muscle guidance process to reach their target apodeme. ed clones in wing discs show cell sorting behavior, causing aggregation and adhesion of only those cells expressing the same complement of cell adhesion molecules. Thus, both reduction and excess of Ed on the 'muscle side' of transient muscle–epidermal adhesions could lead to significant changes in the cell adhesion properties of the developing muscle. The experiments shown in this study for DGripΔ1–3 overexpression in muscle 5 and for VLMs in dgrip mutants imply that a tight balance of DGrip activity might particularly be needed to keep navigating muscle projections motile and to avoid their premature stabilization at ectopic epidermis contacts during the 'steering' process—ultimately instructed by Slit/Robo or other guidance systems. It is likely that Ed and DGrip form complexes enriched at muscle projection membranes to locally control adhesiveness. Ectopic adhesions among muscles cells with aberrant DGrip activity are in fact indicative of changes in muscle adhesiveness (Swan, 2006).
Natural variants of mGRIP missing PDZDs 1–3 have been localized to mammalian synapses, and it has recently been found that the type 5 metalloproteinase MT5-MMP is recruited by GRIP1/2 to growth-cone filopodia and to both mature and developing synapses, where it proteolyses N-cadherins. GRIP2 was also observed to be a member of a Δ-catenin containing complex. Drosophila Echinoid is known to regulate DE-Cadherin in homeotypic cell–cell junctions. Given these promising indications, it will prove interesting to see whether in the context Grip proteins became famous for synapse assembly -- similar molecular strategies are used by the GRIP protein as those described here in the context of muscle morphogenesis (Swan, 2006).
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
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