In the Drosophila visual system, the color-sensing photoreceptors R7 and R8 project their axons to two distinct layers in the medulla. Loss of the receptor tyrosine phosphatase LAR from R7 photoreceptors causes their axons to terminate prematurely in the R8 layer. This study has identified a null mutation in the Liprin-α (for LAR-interacting protein) gene based on a similar R7 projection defect. Liprin-α physically interacts with the inactive D2 phosphatase domain of LAR, and this domain is also essential for R7 targeting. However, another LAR-dependent function, egg elongation, requires neither Liprin-α nor the LAR D2 domain. Although human and Caenorhabditis elegans Liprin-α proteins have been reported to control the localization of LAR, LAR localizes to focal adhesions in Drosophila S2R+ cells and to photoreceptor growth cones in vivo independently of Liprin-α. In addition, Liprin-α overexpression or loss of function can affect R7 targeting in the complete absence of LAR. Despite its reported role in axonal transport of synaptic vesicle components (Miller, 2005), Liprin-α is not required for the transport of endogenous LAR protein to the growth cones of larval R1–R6 photoreceptors. It is concluded that Liprin-α does not simply act by regulating LAR localization but also has LAR-independent functions (Hofmeyer, 2006). A second publication (Choe, 2006) reports similar results.

The color-sensitive photoreceptors of the Drosophila visual system, R7 and R8, provide a simple system in which to study layer-specific axon targeting. Whereas the outer photoreceptors R1–R6 project their axons to the lamina, R7 and R8 project to the medulla, where R8 terminates in the more superficial M3 layer and R7 in the deeper M6 layer. This targeting occurs in two stages, with both R7 and R8 growth cones pausing in separate temporary layers before proceeding to their final positions. Several genes are known to contribute to the establishment of the R7 and R8 projection pattern. The transcription factor Runt is expressed in R7 and R8, and its misexpression is sufficient to target R2 and R5 to the medulla, suggesting that it controls the choice of optic neuropil. Endogenous expression of the homophilic cell adhesion molecule Capricious (Caps) in R8 or its ectopic expression in R7 directs these photoreceptors to terminate in the Caps-positive M3 layer. The transmembrane cadherin Flamingo (Fmi) is required for R8 targeting, whereas loss of either N-cadherin (Ncad) or one of the receptor protein tyrosine phosphatases (RPTPs), PTP69D or LAR, causes R7 to terminate inappropriately in the R8 layer (Hofmeyer, 2006).

Other functions of LAR include axonal patterning of photoreceptors R1–R6 and embryonic motor neurons, synapse morphogenesis at the larval neuromuscular junction (NMJ), and polarization of actin filaments in the follicle cells surrounding the oocyte, which promotes egg elongation along the anterior-posterior axis. It is unclear how LAR and other RPTPs signal within the cell to induce the cytoskeletal rearrangements that mediate these functions. Trio, a guanine nucleotide exchange factor for Rac, and Enabled (Ena), which regulates actin polymerization, show genetic interactions with LAR in both R7 targeting and motor axon guidance. LAR can dephosphorylate both Ena and its antagonist, the cytoplasmic kinase Abelson (Abl). Yeast two-hybrid screens for proteins that bind to the LAR intracellular domain identified both the human and Drosophila homologues of Liprin-α, a protein with an N-terminal coiled-coil domain and a C-terminal LAR-binding liprin homology domain (LHD) consisting of three sterile alpha motif domains (Kaufmann, 2002; Serra-Pages, 1995). Drosophila Liprin-α mutations have the same effects as LAR mutations on NMJ synapse morphology (Kaufmann, 2002), suggesting that the two proteins act together (Hofmeyer, 2006).

Several studies suggest a role for Liprin-α in protein localization. Synaptic vesicle proteins such as synaptotagmin and synaptobrevin are mislocalized in neurons mutant for either Drosophila Liprin-α or the Caenorhabditis elegans Liprin-α homologue syd-2 (Miller, 2005, Zhen, 1999). In Drosophila, this phenotype reflects a requirement for Liprin-α in axonal transport of synaptic vesicles through its binding to the Kinesin-1 (Khc) motor protein (Miller, 2005). In cultured hippocampal neurons, Liprin-α promotes glutamate receptor targeting to synapses (Ko, 2003a, Wyszynski, 2002). The observations that human Liprin-α2 promotes clustering of LAR within the plasma membrane in COS cells, and that SYD-2 and the C. elegans LAR homologue PTP-3 regulate each other’s localization along the nerve cord, suggest that localization also may be the mechanism by which Liprin-α influences LAR function (Hofmeyer, 2006).

This study identifies a likely null mutation in Drosophila Liprin-α that has the same effect on R7 photoreceptor targeting as LAR, yet does not affect two other LAR-dependent processes, egg elongation and motor axon guidance. Liprin-α physically interacts with LAR in embryos and cultured cells; however, the results do not support a primary role for Liprin-α in LAR localization. LAR localization to focal adhesions in cultured Drosophila cells and to photoreceptor growth cones in vivo is independent of Liprin-α. In addition, Liprin-α overexpression can partially restore R7 targeting, and removal of Liprin-α can further reduce R7 targeting in the complete absence of LAR, indicating that some functions of Liprin-α are independent of LAR (Hofmeyer, 2006).

Thus Liprin-α is specifically required in the R7 photoreceptor for its axonal targeting to the correct layer in the medulla. R7 terminates inappropriately in the R8 target layer in the absence of either Liprin-α, LAR, or the D2 domain of LAR to which Liprin-α binds. Liprin-α and LAR both localize to photoreceptor growth cones and are found in the same protein complex. These results imply that a protein–protein interaction between LAR and Liprin-α is important for R7 targeting. The data suggest that this interaction primarily contributes to signaling downstream of LAR, rather than mediating localization of the LAR protein. However, Liprin-α also appears to act in a parallel, LAR-independent pathway (Hofmeyer, 2006).

The C. elegans Liprin-α homologue, syd-2, was identified through its effects on the localization of presynaptic proteins (Zhen, 1999). Previous studies also have shown that in mammalian cells, LAR and Liprin-α colocalize at focal adhesion contacts (Serra-Pages, 1995), but in the absence of cotransfected Liprin-α, LAR is uniformly distributed throughout the plasma membrane (Serra-Pages, 1998). In contrast to these results, no effect of Liprin-α on LAR localization was detected. Despite the reported role for Liprin-α in axonal transport in motor neurons (Miller, 2005), endogenous LAR is correctly transported to the R1–R6 growth cones, and epitope-tagged LAR does not alter its distribution in R7 terminals in Liprin-α mutants. In S2R+ cells, LAR localizes to focal adhesions in the absence of cotransfected or detectable endogenous Liprin-α. It is unlikely that one of the other two Liprin family members encoded in the Drosophila genome can substitute for Liprin-α in LAR localization, because CG10743 is most homologous to human Liprin-β, which does not bind directly to LAR, and CG11206 is not expressed in S2R+ cells (Hofmeyer, 2006).

Because Liprin-α is not required for LAR localization but binds to LAR and has the same effect as LAR on R7 targeting, it may contribute to LAR signal transduction. Although the Liprin-α protein contains no predicted catalytic domains, human Liprin-α1 has been shown to undergo autophosphorylation (Serra-Pages, 2005), suggesting that it might act as a kinase. Alternatively, Liprin-α might promote the association of LAR with its substrates; mammalian Liprin-α family members have been shown to bind to a variety of synaptic molecules (Ko, 2003a; Wyszynski, 2002). Although the LAR substrate Ena binds directly to the intracellular domain of LAR, the Drosophila Trio homologue lacks the domains that mediate LAR binding by human Trio and might require an adaptor protein such as Liprin-α. Liprin-α also might promote interaction of LAR with a cadherin/catenin complex, because β-catenin is a substrate for human LAR in COS cells. Finally, Liprin-α might directly regulate the phosphatase activity of LAR. The D2 domain of LAR and other RPTPs can inhibit their phosphatase activity, in some cases by promoting dimerization. Although the LAR intracellular domain crystallizes as a monomer, homotypic interactions between the LAR transmembrane and intracellular domains have been observed in biochemical assays. Binding of Liprin-α to LAR-D2 might reduce its ability to dimerize (Hofmeyer, 2006).

LAR does not require Liprin-α for all of its functions. The R7 mistargeting phenotype is stronger in LAR mutants than in Liprin-α^oos mutants, although the Liprin-α^oos allele is likely to be a null. More dramatically, egg elongation and ISNb motor axon guidance are unaffected in Liprin-α mutants but strongly affected in LAR mutants. Conversely, removal or overexpression of Liprin-α can alter the extent of correct R7 targeting even in the complete absence of LAR. These observations indicate that Liprin-α does not function exclusively as an adaptor for LAR or a regulator of LAR activity but can also act in a parallel pathway, perhaps by localizing other proteins necessary for R7 targeting. Upstream input for this parallel function might come from Ncad or the RPTP PTP69D, because mutations in either show a LAR-like R7 targeting defect in the adult. However, Liprin-α could mediate only a subset of the functions of these molecules, because Ncad is required at an earlier stage and Ptp69D mutants cause an R1–R6 mistargeting phenotype. LAR and PTP69D might have substrates in common that control R7 targeting, because a chimeric protein with the extracellular domain of LAR and the intracellular domain of PTP69D can rescue R7 targeting in LAR mutants. Taken together, these results show that the simple model that Liprin-α acts by localizing LAR to the appropriate region of the plasma membrane (Serra-Pages, 1998) is unlikely to be correct. Understanding the mechanism of Liprin-α function will lead to new insights into RPTP signaling and layer-specific axonal targeting (Hofmeyer, 2006).

GENE STRUCTURE

cDNA clone length - 4463 bp (transcript A)

Bases in 5' UTR - 199

Exons - 15

Bases in 3' UTR - 658

PROTEIN STRUCTURE

Amino Acids - 1201

Structural Domains

The structural motifs in Liprin-α family members consist of an N-terminal coiled-coil domain, shown to multimerize the protein and a C-terminal LAR binding region (Serra-PagĖs, 1995). While Drosophila liprin-α is 47% identical to mammalian Liprin-α overall, much higher levels of conservation are seen in individual domains. For example, a region of 70 amino acids near the N terminus of Drosphila liprin-α is over 90% identical to mLiprin-α. In addition, the LAR binding domain of Drosophila liprin-α recovered in a protein interaction screen is 75% identical to mLiprin-α and 68% identical to its C. elegans homolog (Kaufmann, 2002).

date revised: 19 January 2007

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