To understand the functional significance of the GUK domain of Dlg, binding partners of this domain were sought using a yeast two-hybrid screen. The GUK domain of Dlg (amino acids 765-960 was used as bait to screen a late embryonic stage Drosophila cDNA library. Thirty-eight interacting clones were recovered from this screen, and from these, nine were overlapping cDNAs representing a single novel gene, GUK-holder (Mathew, 2002).
To determine the precise regions of interaction between Dlg and GUKh, deletion constructs of the Dlg GUK domain and of the GUKh C terminus were generated and assayed for binding using the yeast two-hybrid assay. Nearly the entire GUK domain is necessary for an interaction with GUKh, since deletion of more than 15 residues from either end results in a loss of binding. A construct encompassing the last 156 amino acids of GUKh (amino acids 888-1044) is sufficient to mediate binding to the GUK domain of Dlg, defining this region as the GUK-holding domain (Mathew, 2002).
An affinity-purified polyclonal antiserum directed against the last 238 C-terminal amino acids of GUKh (GUKh-C) was prepared. In Western blots from body wall muscle extracts, the antibody detects a single band of ~110 kDa, consistent with the predicted size of the L isoform of GUKh. Moreover, anti-GUKh immunoreactivity is reduced at the NMJs or CNS of the hypomorphic gukh mutants gukhJ3E1and gukh2, eliminated in gukh2EM9 embryos, and enhanced upon overexpression of a gukh transgene, confirming the specificity of the antibody (Mathew, 2002).
To establish that Dlg and GUKh interact in vivo, a coimmunoprecipitation of body wall muscle extracts was performed using GUKh antibody. In wild-type, Dlg-specific bands at 97 and 116 kDa coprecipitate with the 110 kDa GUKh band, suggesting that GUKh exists in the same complex as Dlg. In contrast, Dlg is not coimmunoprecipitated from dlgXI-2 mutants that lack the GUK domain. Together, these results strongly suggest that GUKh binds to the GUK domain of Dlg in vivo (Mathew, 2002).
The interaction of Dlg and GUKh is reminiscent of the interaction between the GUK domain of mammalian MAGUKs and GKAP (Kim, 1997; Takeuchi, 1997). Moreover, while GUKh and GKAP do not share significant sequence homology, both proteins terminate in a similar tS/TXV/L/I PDZ binding motif (i.e., tETAL versus tQTRL. In fact, GKAP proteins link the GUK domain of PSD-95/SAP90 to the PDZ domain of Shank/ProSAP (Boeckers, 1999; Tu, 1999). By analogy, it is inferred that GUKh might link Dlg to other PDZ domain-containing proteins. Recent studies have revealed that at epithelia Dlg exists in a complex with Scribble (Scrib), a protein comprising 16 leucine-rich repeats followed by four PDZ domains. However, the molecular nature of this interaction remained elusive. In this study, a coimmunoprecipitation assay was performed on body wall muscle extracts using a Scrib-specific antibody. Anti-Scrib efficiently coimmunoprecipitates Dlg from wild-type but not from a severe hypomorphic scrib allele. This indicates that, similar to the case in epithelia, Dlg and Scrib may exist in a complex at the NMJ. In line with this finding, Scrib exhibits striking colocalization with Dlg at type I boutons (Mathew, 2002).
Whether GUKh might provide a physical link between Dlg and Scrib was assessed. Indeed, GUKh is detected in anti-Scrib immunoprecipitates from wild-type but not from scrib mutant extracts. Moreover, immunoprecipitation of Dlg by anti-Scrib antibodies from a hypomorphic gukh allele is dramatically reduced (Mathew, 2002).
To investigate the possibility that the interaction between GUKh and Scrib might be direct, a yeast two-hybrid assay was used; this shows that GUKh specifically interacts with the PDZ2 domain of Scrib but not with either its PDZ3-4 or its LRR motifs. The interaction between GUKh and the PDZ2 domain of Scrib is mediated by the C terminus of GUKh, since just the ten last amino acids of GUKh are sufficient for this interaction. Deletion of the last 23 amino acids of GUKh (GUKh-DeltaC) prevents the interaction with the PDZ2 domain of Scrib. Moreover, when the ten amino acid peptide contains a mutation (L->A) at the C-terminal residue, it fails to interact with PDZ2. In addition, the last ten amino acids of Shaker K+ channel, which strongly binds to PDZ1-2 of Dlg, failed to bind PDZ2 of Scrib, demonstrating a degree of ligand specificity. In contrast, constructs encompassing PDZ1-2 or PDZ3 of Dlg failed to bind GUKh. Together, the localization, immunoprecipitation, and yeast two-hybrid studies strongly suggest that Dlg, GUKh, and Scrib may form a tripartite complex in which GUKh serves as a physical link between Dlg and Scrib (Mathew, 2002).
Communication between cortical cell polarity cues and the mitotic spindle ensures proper orientation of cell divisions within complex tissues. Defects in mitotic spindle positioning have been linked to various developmental disorders and have recently emerged as a potential contributor to tumorigenesis. Despite the importance of this process to human health, the molecular mechanisms that regulate spindle orientation are not fully understood. Moreover, it remains unclear how diverse cortical polarity complexes might cooperate to influence spindle positioning. Spindle orientation roles have been identified for Dishevelled (Dsh), a key regulator of planar cell polarity, and Discs large (Dlg), a conserved apico-basal cell polarity regulator, effects which were previously thought to operate within distinct molecular pathways. This study identified a novel direct interaction between the Dsh-PDZ domain and the alternatively spliced 'I3-insert' of the Dlg-Hook domain, thus establishing a potential convergent Dsh/Dlg pathway. Furthermore, a Dlg sequence motif that is necessary for the Dsh interaction was identified that shares homology to the site of Dsh binding in the Frizzled receptor. Expression of Dsh enhanced Dlg-mediated spindle positioning similar to deletion of the Hook domain. This Dsh-mediated activation was dependent on the Dlg-binding partner, GukHolder (GukH). These results suggest that Dsh binding may regulate core interdomain conformational dynamics previously described for Dlg. Together, these results identify Dlg as an effector of Dsh signaling and demonstrate a Dsh-mediated mechanism for the activation of Dlg/GukH-dependent spindle positioning. Cooperation between these two evolutionarily-conserved cell polarity pathways could have important implications to both the development and maintenance of tissue homeostasis in animals (Garcia, 2014: PubMed).
The question of a possible interaction between Dlg and GUKh at synaptic sites was addressed by examining the colocalization of the proteins through development. GUKh makes its first appearance presynaptically at the NMJ during embryonic stage 17 where it overlaps with the neuronal marker, anti-HRP, and with Dlg. At this time, GUKh is distributed throughout the developing boutons. During the first larval instar, the protein becomes enriched at the rim of the boutons in colocalization with Dlg. This pattern is maintained through late larval development. Interestingly, a similar developmental pattern of expression is observed for Dlg. However, while Dlg immunoreactivity is found throughout a large extent of the postsynaptic junctional region (SSR), typically, GUKH immunoreactivity is distributed in interrupted patches along the synapse border that usually extend a short way into the bouton interior. These observations together with the protein interaction studies provide strong evidence for a direct interaction between GUKH and DLG at the NMJ. GUKh immunoreactivity is also found in the embryonic and larval CNS and asymmetrically distributed in neuroblasts (Mathew, 2002).
Comparison of GUKh and Dlg distribution at synaptic boutons has revealed that, although both proteins colocalize at bouton borders, they also show distinctly complementary patterns during bouton budding. Larval NMJs expand during development to compensate for an increase in muscle size. This expansion involves an enhancement in bouton and active zone number, which serves to maintain synaptic strength despite the changes in muscle size. The process of NMJ expansion occurs by the formation of new boutons that bud off from existing boutons, as has been described both in vivo and in fixed NMJs (Mathew, 2002).
GUKh is enriched at budding boutons where it fills the entire bud, in contrast to its more peripheral distribution in the mature boutons. To compare the distribution of GUKh and Dlg during bouton budding, complete confocal Z series of synaptic boutons was acquired and their expression was analyzed in single slices. In first instar larva GUKh and Dlg expression the distribution of both proteins changed at different stages of bouton budding, consistent with a strikingly dynamic expression.
During the stage of protrusion, GUKh is highly enriched in the core of the protruding bud. At the same stage, Dlg immunoreactivity decreases at the site of protrusion and becomes strong at the borders immediately adjacent to the site of low Dlg. Throughout this stage, GUKh and Dlg colocalize at the bouton border, except for the leading edge of the protrusion, where Dlg is low (Mathew, 2002).
Once the bud separates from the parent bouton, GUKh remains enriched in the bud but disappears from the neck of the bud. In contrast, Dlg completely disappears from the distal border of the bud and becomes highly enriched at the neck of the bud. The bud takes on a distinctly bouton-like morphology, and in concert, the distribution of GUKh and Dlg is similar to a mature bouton, i.e., both proteins localize at the periphery of the bouton. However, GUKh is still substantially enriched at the distal border of the nascent bouton. Similar observations were made in NMJ from older larva; thus, GUKh and Dlg appear to be dynamically localized during bouton budding, overlapping at the edges but being complementary at the buds (Mathew, 2002).
The gukh gene maps to position 91E on the right arm of the third chromosome. Within this region, two homozygous viable P element insertions subsequently referred to as gukhJ3 and gukh2 were identified that exhibit a moderate but significant decrease in GUKh immunoreactivity at larval NMJs or CNS. Using inverse PCR, it was determined that in gukhJ3 and gukh2 the P elements are inserted ~60 kb and 380 bp, respectively, upstream from the transcriptional start site of gukh. Several genes are predicted to lie between the P insertion in gukhJ3 and the first exon of gukh. It is suggested that both P insertions affect regulatory elements required for proper gukh expression. To test whether the P insertions are responsible for the reduction of GUKh immunoreactivity, additional alleles by P element excision were generated. One new allele, gukhJ3E1, resulted from an imprecise excision which caused a deletion of ~5 kb. Both homozygous and hemizygous gukhJ3E1 flies exhibited decreased viability and, most notably, a further reduction in GUKh immunoreactivity as compared to gukhJ3. In contrast, synaptic GUKh immunoreactivity was reverted to wild-type levels in another allele, gukhrev, in which the P element was excised precisely. In the case of gukh2, imprecise excision of the P element results in complete elimination of GUKh immunoreactivity in the embryo, but this mutation is lethal prior to hatching (Mathew, 2002).
To understand the role of GUKh at synapses, the morphology of gukhJ3, gukhJ3E1, gukhJ3E1/Df(3R)Cha7, and gukh2 NMJs was examined. No noticeable defects in synaptic bouton number and morphology were found upon examining preparations stained with the presynaptic marker anti-HRP. Similarly, immunocytochemical analysis of the distribution of several synaptic proteins, including FasII, Dlg, Synapsin, Cysteine string protein (CSP), and Synaptotagmin and CaMKII revealed no significant changes in their distribution in gukh mutants (Mathew, 2002).
In contrast, dramatic changes in the synaptic distribution of Scrib were observed in gukh mutants. In wild-type larvae, Scrib tightly colocalizes with Dlg at type I boutons. Interestingly, Scrib immunoreactivity is much less intense, appearing dramatically mislocalized or not as tightly concentrated at the rim of type I boutons in gukhJ3E1 homozygotes, in gukhJ3E1/Df, and in gukh2. The decrease in synaptic Scrib localization in both the P element insertion allele (gukhJ3) and in the more severe excision allele (gukhJ3E1) was specific, as targeted expression of a UAS-gukh-c transgene rescued the mislocalization of Scrib, and synaptic Scrib localization was restored in gukhrev and gukh2revEM30 (Mathew, 2002).
To determine if GUKh is required pre- or post-synaptically to maintain normal Scrib localization at synaptic boutons, Gal4 driver BG487 was used to target muscle-specific GUKh-C expression and C380 to drive transgenic expression in the motorneurons. The UAS-gukh-c transgene encodes an amino-terminally truncated variant of GUKh (aa 652-1044), which lacks the WH1 domain but still contains the Dlg and Scrib binding motifs. As indicated by increased immunoreactivity, GUKh-C becomes localized to type I boutons upon both pre- and post-synaptic expression. Surprisingly, driving GUKh-C in motorneurons is sufficient to rescue the abnormal Scrib localization in gukhJ3E1 mutants. However, driving GUKh-C in the muscles alone is much less effective in rescuing Scrib localization at type I boutons. The reduced rescue observed with postsynaptic expression might be due to the lack of the amino-terminal region of the transgene (Mathew, 2002).
It was next determined whether mutations in dlg affect the synaptic localization of Scrib or GUKh. In dlgX1-2 mutants, Scrib is mislocalized to an extent similar to that observed in gukh mutants, and this effect is enhanced in dlg;gukh double mutants. Thus, both Dlg and GUKh are required for normal Scrib localization at NMJs. This relationship is unidirectional, since both Dlg and GUKh immunoreactivities remain unaltered at NMJs in scrib2 mutant larvae. A simple explanation for the mislocalization of Scrib in dlgX1-2 mutants would be that Dlg recruits GUKh to the NMJ. However, it was found that GUKh immunoreactivity is normal in dlgX1-2 mutants (Mathew, 2002).
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date revised: 10 April 2015
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