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The extreme 3’, carboxy end of the APC gene product contains the S/TXV motif (S/T, serine or threonine; X any amino acid; V, valine), which can interact with the PDZ domain of hDLG (the human homologue of Drosophila lethal (1) discs large - 1 tumour suppressor gene) - Matsumine et al. (1996). APC interaction with hDLG negatively

regulates cell cycle progression from G0/G1 to S phase (Ishidate et al. 2000).

Disruption of this effect may contribute to the pathogenicity of truncated APC mutants because APC S/TXV motif loss is nearly universal in APC mutants, so far described.

hDLG contains three PDZ domains. PDZ domain containing proteins (family members: hDLG, PSD-95, PSD-93, SAP 102, ZO-land ZO-2 (Craven and Bredt 1998) have been shown to associate with membrane bound proteins to induce protein clustering. PDZ domains are a target of proteins associated with tumorigensis (human papilloma virus E6 oncoprotein, adenovirus E4 oncoprotein and human T cell leukaemia virus type 1 Tax protein) - (Kiyono, et al. 1997 and Lee et al. 2000). These data suggest that disruption of the APC-hDLG interaction could contribute to cancer progression.

Native APC can block cell cycle (Baeg et al. 1995) by inhibiting the transition from G0/G1 to S phase. Some APC mutations at the extreme 3’ end of the gene appear to

be p-catenin degradation competent (implying in vivo mutant protein stability and normal intracellular APC dosage) and only lack features such as the hDLG binding motif (Miyoshi et al. 1992, Pedemonte et al. 1998 Korinek et al. 1997, Morin et al. 1997). Cell culture mutant hDLG transfection experiments have demonstrated that hDLG inhibits the transition from G0/G1 to S phase of the cell cycle, i.e. blocking cell cycle. Mutant hDLG (which fails to block cell cycle) partially inhibits the ability of APC to block cell cycle. APC mutants fail to block cell cycle, they also lack the three carboxy- terminal TSV amino-acids (i.e. hDLG binding site). Therefore, hDLG may be an effector pathway for some of APC’s cell cycle blocking activities.

A clue to hDLG’s normal function comes from the study of Drosophila neuro-muscular junctions (NMJs) - Craven and Bredt (1998). DLG mutants develop normal NMJs in early lava I stages but these fail to develop properly as the organism grows. A possible reason is that DLG clusters Fasciclin II, which then stabilises the inter-cellular junctions (synapses). Protein clustering at specialised cell membrane sites may be critical for

effective signal transduction and cell function (e.g. at a synapse or adherens junction). Perhaps this is similar to the APC-hDLG-E-cadherin situation, the carboxy-terminus of APC aggregating molecules involved at the tight and zona-occludens junctions. This could stabilise the intercellular junctions between epithelial cells and help the cell

achieve apical-luminal polarisation. Whereas in the neuron, stable synapses are required for proper neuro-epithelial function, in the colonic epithelium lateral growth inhibitory messages are mediated by stable adherens and tight junctions. APCs/hDLG role at intercellular junctions may therefore simply be the physical linking of

cytoskeletal (the plus end of the microtubule) and junctional molecules, thus producing epithelial stabilisation.

Structure analysis of hDLG suggests that it may exert a major action as a cross-linking molecule. Multiple PDZ domain containing proteins may form a ‘backbone’ that links together functionally related proteins. Disruption of the backbone could disrupt function by dispersal of proteins, which need spatial colocalisation to function optimally. Work studying neuro-epithelial Shaker-type K+ channels confirms the interaction between PDZ bearing transmembrane proteins and members of the PSD family (e.g. hDLG). hDLG contains three PDZ binding sites that interact with carboxy tails of

transmembrane proteins. hDLG and other PSD containing proteins induce protein clustering in a diverse range of tissues. The protein clusters have specialised function, e.g. neurotransmitter communication at the synapse (Kim et al. 1995, Kornau et al. 1995). Furthermore hDLG is localised to the lateral cell borders of colonic epithelial cells (basal to the adherens junctions) and APC spatially colocalises at the cell membrane with hDLG.

To complicate matters futher, hDLG is not the only candidate PDZ containing gene in the vicinity of APC at the cell membrane. Close to the adherens junction in the colonic epithelial cell is the zona occludens (the tight junction, which ‘waterproofs' intestinal epithelia). Two molecules ZO-1 and ZO-2 both constitute part of the epithelial cell tight

junction and have high homology to hDLG (ZO-1 contains the same number of PDZ domains). Therefore, they may also bind the APC carboxy- terminal. The original identification of hDLG as an APC partner protein identified a protein sequence that spanned hDLG amino acids 199 to 507. The sequence contains two and half of the PDZ domains common to this family of molecules (Kim et al. 1995). Therefore, it is possible that proteins which share the same number of PDZ domains, which localise to the cell membrane and are present in colonic epithelium, could also interact with APC. Furthermore the original work of Matsumine et al. (1996) used a human brain cDNA library for the two-hybrid screen and an embryonic mouse brain lysate for their in vivo binding studies. The detection of APC protein partners could have been influenced by patterns of gene expression in these systems.

The final twist in the APC-hDLG tale comes from consideration of APC as a dimer. If, APC forms homotypic amino-terminus interactions it could reasonably be considered a dimer. However since hDLG contains multiple PDZ binding domains it could

conceivably cause the cross-linking of APC at the cell membrane, generating a much larger protein complex, i.e. a tandem complex is generated because of amino- and carboxy- interactions. This suggests a mechanism for extreme 3’ mutations to disrupt APC function. APC mutations that are produced by extreme 3’ mutations may cause WNT signal transduction instability. Partial failure of APC localisation due to

insufficient cross-linking might allow un-phosphorlyated p-catenin to ‘leak’ into the

nucleus because local epithelial cell junction APC concentrations are insufficient to

regulate p-catenin fully, i.e. there is local insufficency of APC at the cell membrane, cf. global insufficiency of APC secondary to hemizygosity, NMD etc.