BIOLOGICAL OVERVIEW

Membrane-associated guanylate kinases (MAGUKs), such as Discs-large (Dlg), play critical roles in synapse maturation by regulating the assembly of synaptic multiprotein complexes. Previous studies have revealed a genetic interaction between Dlg and another PDZ scaffolding protein, Scribble (Scrib), during the establishment of cell polarity in developing epithelia. The biochemical nature of this interaction has remained elusive, raising questions regarding the mechanisms by which the actions of both proteins are coordinated. This study reports a new Dlg-interacting protein, GUK-holder (GUKh), that interacts with the GUK domain of Dlg and that is dynamically expressed during synaptic bouton budding. At Drosophila synapses Dlg colocalizes with Scrib and this colocalization is likely to be mediated by direct interactions between GUKh and the PDZ2 domain of Scrib. Dlg, GUKh, and Scrib form a tripartite complex at synapses, in which Dlg and GUKh are required for the proper synaptic localization of Scrib. These results provide a mechanism by which developmentally important PDZ-mediated complexes are associated at the synapse (Mathew, 2002).

While the PDZ and SH3 domains of MAGUKs are known to bind components required for synapse function, the significance of the guanylate kinase-like (GUK) domain has remained puzzling. Several studies suggest that it might act as a protein interaction domain. For example, in mammals, this domain binds to GKAP/SAPAPs (Kim, 1997; Takeuchi, 1997), which are in turn linked to Shank/ProSAP (Naisbitt 1999; Boeckers, 1999). It has also been reported to bind MAP1A (Brenman, 1998), to a kinesin-like protein (Hanada, 2000), to SPAR, an actin cytoskeleton regulator (Pak, 2001), and to interact intramolecularly with the SH3 domain (Mathew, 2002 and references therein).

In Drosophila, dlg mutants in which the GUK domain is absent exhibit abnormalities in synapse structure. Moreover, transgenic Dlg lacking the GUK domain fails to localize at synapses when expressed in a dlg mutant background. These findings imply that the GUK domain is required for a synaptic function and targeting of Dlg. To gain further insight on how the GUK domain of DLG exerts its various functions, proteins interacting with this domain were sought. GUK-holder, a novel synaptic protein contains a WH1/EVH1-like domain in its N-terminal half and a PDZ binding motif at its C terminus; the PDZ binding motiif has been identified as a GUK interactor. GUKh is expressed in a dynamic fashion during synaptic bouton formation. In addition, it also binds to a PDZ domain of Scribble (Scrib; a tumor suppressor protein interacts genetically with Dlg in developing epithelia) thus physically linking Dlg to Scrib. Indeed, coimmunoprecipitation analyses together with immunocytochemical studies on wild-type and mutant larvae provide strong evidence that Dlg, GUKh, and Scrib exist in a tripartite complex at the NMJ. Most notably, normal GUKh function is required for the synaptic localization of Scrib (Mathew, 2002).

Together, these yeast two-hybrid, coimmunoprecipitation, and colocalization studies provide compelling evidence that GUKh interacts with Dlg in vivo. This interaction is mediated by a region near the C terminus of GUKh. However, as revealed by genetic analysis, the synaptic localization of GUKh does not depend on Dlg. This suggests that domains other than the Dlg interacting motif may mediate its synaptic localization. For instance, the single WH1-like domain of GUKh might interact directly or indirectly with the synaptic cytoskeleton. WH1 domains in other proteins bind F-actin, actin-associated proteins such as zyxin, vinculin, and profilin, or the spectrin-bound scaffolding protein Shank/ProSAP. Association of GUKh with cytoskeletal elements might also be mediated by those sequences that exhibit moderate similarity to the actin binding protein kelch (Mathew, 2002).

The GUK domain of Dlg and related MAGUKs is enzymatically inactive and may have evolved as a protein-protein interaction domain. A number of vertebrate GUK domain binding partners, including GKAP, MAP1A, the kinesin GAKIN, and the Rap-specific GTPase activating protein SPAR, have been identified (Kim, 1997; Brenman, 1998; Hanada, 2000). Although these proteins are structurally quite diverse, a common theme appears to be their association with the cytoskeleton (Mathew, 2002).

While an association of GUKh with the actin-based synaptic cytoskeleton currently remains hypothetical, the C-terminal tETAL motif specifically binds to the second PDZ domain of Scrib. Anatomical and biochemical experiments suggest that in vivo, Dlg, Scrib, and GUKh may exist in the same complex at the NMJ. Alternatively, the three proteins could interact pairwise, forming separate heterodimers. Since GUKh was found to still localize normally at dlg mutant NMJs, it is proposed that Dlg and GUKh act in concert rather than in a hierarchical manner to recruit Scrib. As a possible mechanism, binding to the GUK domain of Dlg could cause sterical changes in GUKh, such that the tETAL motif becomes available for interaction with Scrib. A caveat to this study is that hypomorphic gukh mutants were used, and therefore, a requirement of GUKh in Dlg localization cannot be ruled out (Mathew, 2002).

The presence of multiple protein-protein interaction domains in both Dlg and Scrib suggests that GUKh may link two different multiprotein complexes in a defined spatial arrangement. This is reminiscent of the coupling of NMDA receptors and metabotropic glutamate receptors at mammalian PSDs through a quaternary complex (Tu, 1999) formed by PSD-95, Homer (see Drosophila Homer), GKAP and Shank/ProSAP (Mathew, 2002).

While Dlg and Scrib are colocalized along the rims of synaptic boutons, which, as has been demonstrated for Dlg, comprise both the presynaptic membrane and the postsynaptic junctional region (SSR), GUKh intersects that region only in a narrow strip. Yet, in budding boutons, GUKh displays a complementary pattern to Dlg. These observations suggest that GUKh may not be continuously bound to Dlg but rather may be involved in transient interactions. The process of bouton budding is a dynamic process that is characterized by equally dynamic changes in both GUKh and Dlg distribution. The accumulation of GUKh at the core of budding boutons and the disappearance of Dlg at the border of buds suggest that both proteins serve different roles during this process. Interestingly, FasII, a molecule that mediates synapse stabilization but that also imposes an adhesive constraint on synaptic growth, faithfully resembles the changes in distribution of Dlg during budding, consistent with a role for Dlg in synaptic localization. The presence of GUKh at budding regions may represent a role for this protein in destabilizing regions of the synaptic bouton, thereby allowing for bud formation (Mathew, 2002).

In contrast to GUKh, Scrib is expressed throughout the SSR in exact colocalization with Dlg. Nonetheless, Scrib localization at distal regions of the SSR is also affected in gukh mutants. In fact, considering the hypomorphic character of the gukh alleles that were used in this study, the effect on Scrib localization appears remarkably strong. This observation might indicate that GUKh activity is required only temporarily and/or in a locally restricted fashion to prime a secondary mechanism by which Scrib becomes associated with the SSR, e.g., through a more direct interaction with Dlg. Interestingly, presynaptic expression of GUKh-C is largely sufficient to restore postsynaptic Scrib localization at gukh mutant NMJs. Together, these observations suggest a second, more indirect mechanism by which GUKh contributes to the recruitment of Scrib to the postsynaptic SSR; such a mechanism may involve trans-synaptic signaling (Mathew, 2002).

These studies provide evidence for one mechanism by which scaffolding proteins with different interaction domains may be linked to form a network of multiprotein complexes. GUKh, in physically linking Dlg and Scrib, can therefore bring together these complexes and their associated proteins. Since a single protein forms this link, it would be a straightforward point at which to also separate the complexes, along with their actions, to regulate different aspects of synapse formation. Examples would be during synapse stabilization and during synapse growth through bouton budding. Thus, this work provides a means by which macromolecular complexes can mediate and finely tune various structural changes at the highly dynamic structure of the synapse (Mathew, 2002).

PROTEIN STRUCTURE

Amino Acids - 1044 and 534

Structural Domains

To characterize the gukh transcription unit, a database analysis was performed and several overlapping expressed sequence tag (EST) clones were identified. Further sequencing of these EST clones and alignment with the genomic region indicated that the gukh transcription unit covers three conceptual genes predicted by the BDGP database (CG5456, CG14288, and CG6003), thereby comprising at least six exons spread over a 38 Kb region. The deduced protein sequence comprises 1044 residues, with a calculated molecular weight of 111.4 kDa (long isoform, 'L'). In addition, some EST clones represent an alternatively spliced transcript missing the fifth exon, suggesting the existence of a C-terminally truncated isoform (short isoform, 'S'; 534 amino acids, 57.6 kDa) (Mathew, 2002).

The predicted GUKh protein exhibits no signal sequences or transmembrane domains, consistent with it being intracellular. A homology search revealed a region with similarity to the WH1/EVH1 domain of the Drosophila homolog of Suppressor of cAR (SCAR; 32% identity; 54% similarity: see Drosophila SCAR) and its murine ortholog WAVE-1. Moreover, a region of moderate homology to the Drosophila actin binding protein Kelch is found within the C-terminal half of GUKh. This region of GUKh also includes a predicted PEST sequence. The Dlg binding region of GUKh maps to the C-terminal third of the protein, as deduced from the overlapping cDNAs obtained from the yeast two-hybrid screen. Notably, GUKh terminates in the potential PDZ binding motif tETAL (Mathew, 2002).

Evolutionary Homologys

Components of the planar cell polarity (PCP) pathway are required for the caudal tangential migration of facial branchiomotor (FBM) neurons, but how PCP signaling regulates this migration is not understood. In a forward genetic screen, a new gene was identified, nhsl1b, that is required for FBM neuron migration. nhsl1b encodes a WAVE-homology domain-containing protein related to human Nance-Horan syndrome (NHS) protein and Drosophila GUK-holder (Gukh), which have been shown to interact with components of the WAVE regulatory complex that controls cytoskeletal dynamics and with the polarity protein Scribble, respectively. Nhsl1b localizes to FBM neuron membrane protrusions and interacts physically and genetically with Scrib to control FBM neuron migration. Using chimeric analysis, it was shown that FBM neurons have two modes of migration: one involving interactions between the neurons and their planar-polarized environment, and an alternative, collective mode involving interactions between the neurons themselves. The first mode of migration requires the cell-autonomous functions of Nhsl1b and the PCP components Scrib and Vangl2 in addition to the non-autonomous functions of Scrib and Vangl2, which serve to polarize the epithelial cells in the environment of the migrating neurons. These results define a role for Nhsl1b as a neuronal effector of PCP signaling and indicate that proper FBM neuron migration is directly controlled by PCP signaling between the epithelium and the migrating neurons (Walsh, 2011).

date revised: 25 September 2023

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