The receptor protein tyrosine phosphatase LARpromotes R7 photoreceptor axon targeting by aphosphatase-independent signaling mechanismKerstin Hofmeyer and Jessica E. Treisman1

Kimmel Center for Biology and Medicine of the Skirball Institute and Department of Cell Biology, New York University School of Medicine,540 First Avenue, New York, NY 10016

Edited by Trudi Schupbach, Princeton University, Princeton, NJ, and approved September 30, 2009 (received for review April 10, 2009)

Receptor protein tyrosine phosphatases (RPTPs) control manyaspects of nervous system development. At the Drosophila neu-romuscular junction (NMJ), regulation of synapse growth andmaturation by the RPTP LAR depends on catalytic phosphataseactivity and on the extracellular ligands Syndecan and Dally-like.We show here that the function of LAR in controlling R7 photo-receptor axon targeting in the visual system differs in severalrespects. The extracellular domain of LAR important for this pro-cess is distinct from the domains known to bind Syndecan andDally-like, suggesting the involvement of a different ligand. R7targeting does not require LAR phosphatase activity, but insteaddepends on the phosphatase activity of another RPTP, PTP69D. Inaddition, a mutation that prevents dimerization of the intracellulardomain of LAR interferes with its ability to promote R7 targeting,although it does not disrupt phosphatase activity or neuromuscu-lar synapse growth. We propose that LAR function in R7 is inde-pendent of its phosphatase activity, but requires structural fea-tures that allow dimerization and may promote the assembly ofdownstream effectors.

synapse � neuromuscular junctions � dimerization � wedge

Receptor protein tyrosine phosphatases (RPTPs) are re-quired for nervous system development in both vertebrates

and invertebrates (1). Of the six Drosophila RPTPs, Leukocyteantigen-related (LAR) has been studied in most detail due to itsnon-redundant role in several developmental processes. In Larmutant embryos, motor neurons in the intersegmental nerve b(ISNb) fail to innervate the appropriate muscles and aberrantlytrack along the ISN (2). LAR has two distinct functions at thesynapses formed by larval motor neurons on their target muscles.Synapse size as defined by the number of synaptic boutonspresent at these larval neuromuscular junctions (NMJs) isproportional to Lar dosage; and LAR controls active zonemorphogenesis and thus synaptic strength (3). In the visualsystem, LAR enables photoreceptor axons to establish connec-tions to the correct synaptic partners. Photoreceptors R1–R6project into the lamina, where the axons from a single omma-tidium defasciculate and connect to six different laminar car-tridges; this defasciculation requires Lar (4). Photoreceptors R7and R8, which mediate color vision, project beyond the laminato terminate in two distinct layers of the medulla, R8 in M3 andR7 in the deeper M6 layer (5). In Lar mutants, most R7 axonsterminate inappropriately in M3, the same layer as R8 (4, 6).

LAR and its vertebrate homologues PTP� and PTP� are typeIIa RPTPs, which have two intracellular phosphatase domains(D1 and D2) and extracellular Ig (Ig) and fibronectin type III(FNIII) domains. The membrane-distal D2 domains of suchRPTPs show no phosphatase activity on artificial substrates invitro (7–9). Nevertheless, the LAR D2 domain is essential for R7targeting, where it may act by recruiting the scaffolding proteinLiprin-� (10) or regulating the activity of the D1 domain (8). Animportant class of ligands for type IIa RPTPs are heparan sulfateproteoglycans (HSPGs) such as Agrin and Collagen XVIII,

which bind to PTP� (11), and Syndecan (Sdc) and Dally-like(Dlp), which control the activity of Drosophila LAR in motorneurons (12, 13). Sdc and Dlp both bind to the Ig domains ofLAR, but Sdc promotes LAR activity while Dlp antagonizes it(12, 13). Of the nine LAR FNIII domains, only the fifth hasknown binding partners, the Laminin-Nidogen extracellularmatrix complex and a small alternatively spliced secreted isoformof LAR itself (14, 15).

Ligand binding can control RPTP activity by regulatinginteractions between receptor molecules; for example, the cy-tokine Pleiotrophin inhibits the activity of RPTP� by inducingits oligomerization (16). Numerous RPTPs have been shown toform dimers in cultured cells (17–20). The crystal structure of theD1 phosphatase domain of RPTP� shows a dimer in which awedge from the juxtamembrane region of one monomer blocksthe active site of the other monomer (21), providing a possiblemechanism for dimerization-induced inhibition. Mutations inthis wedge reduce RPTP� dimerization, restore activity to formsof the RPTP CD45 or RPTP� forced to dimerize by changes intheir extracellular domains, and increase the activity of CD45 invivo (18, 22–25). Ligand binding may also have effects other thancontrolling phosphatase activity, since there are reports ofphosphatase-independent functions for RPTPs; for instance,PTP� mediates cell adhesion independently of its intracellulardomain (26, 27), and the intracellular domain of ICA512 acts asa transcription factor (28).

We show here that R7 targeting requires the three membrane-proximal FNIII domains of LAR, rather than the Ig domains,suggesting the involvement of a ligand other than Sdc or Dlp.Unlike NMJ growth, R7 targeting does not require catalyticresidues of the phosphatase domains. We also report that amutation in the wedge inhibits dimerization of the LAR intra-cellular domain. This mutant form of LAR retains phosphataseactivity and the ability to promote NMJ growth, but is notfunctional in R7. These results show that LAR function in R7targeting is independent of its phosphatase activity, but requiresa conformation dependent on the wedge domain.

ResultsPhosphatase Activity Is Not Required for LAR Function in R7 Targeting.Although RPTPs are generally thought to act by dephosphory-lating substrates, several studies support the existence of phos-phatase-independent functions (26–28). Phosphatase activity ofLAR is essential for its function in regulating NMJ size (13) andfor its ability to cause motor axon guidance defects when

Author contributions: K.H. and J.E.T. designed research; K.H. performed research; K.H. andJ.E.T. analyzed data; and K.H. and J.E.T. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1To whom correspondence should be addressed. E-mail: jessica.treisman@med.nyu.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0903961106/DCSupplemental.

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ectopically expressed (12), but not to restore viability to Larmutants (8). In the absence of LAR, most R7 photoreceptoraxons terminate at the more superficial R8 layer (4, 6) (Fig. 1A–D). This mistargeting can be strongly rescued by pan-neuronalexpression of wild-type UAS-LAR driven by elav-GAL4 (Fig.1F). To determine whether the function of LAR in R7 targetingrequires its catalytic activity, we tested whether Lar mutantscould be rescued by UAS-LAR transgenes containing pointmutations that change the catalytic cysteine to serine in the D1(LAR-D1CS), the D2 (LAR-D2CS) or both phosphatase do-mains (LAR-2XCS) (Fig. 1 G and H) (8). Surprisingly, trans-genes with either or both cysteines mutated strongly rescued R7targeting in a Lar null mutant background; indeed, phosphatase-dead LAR-D1CS was as active as wild-type LAR in this assay(Fig. 1F). Nevertheless, the cytoplasmic domain of LAR isessential for R7 targeting (6, 10), indicating that LAR is notsimply an extracellular adhesion molecule, but has an intracel-lular function.

Mutating the catalytic cysteine might create a substrate-trapping form of the enzyme, which could bind the substrate inits active site without dephosphorylating and releasing it (29, 30).By removing phosphorylated substrate from circulation, such asubstrate-trap could mimic the function of the wild-type enzyme.To test this hypothesis, we mutated a conserved tyrosine residuein the phosphotyrosine recognition loop, which interacts with

phosphotyrosine to stabilize substrate binding in the active site(31, 32). The presence of a leucine at this position in the LARD2 domain (Fig. 1G) is one of the two amino acid substitutionsthat render the domain catalytically inactive (33). To preventprolonged substrate binding in the active site of LAR-D1CS, wechanged the tyrosine in the LAR D1 domain (Y1504) to leucine.However, this UAS-LAR-D1YLCS transgene (Fig. 1 G and H)still strongly rescued R7 targeting in Lar mutants (Fig. 1 E andF), suggesting that LAR-D1CS promotes R7 targeting througha mechanism other than substrate sequestration.

The LAR-D1CS protein used in this and previous studies wasshown to lack all phosphatase activity in vitro on an artificialsubstrate (8). We wanted to test its activity on a substraterelevant for LAR function in vivo. The intracellular LAR-binding protein Liprin-� acts downstream of LAR in NMJsynapse growth and photoreceptor axon targeting, and is ty-rosine phosphorylated in S2R� cells (3, 10, 34). To facilitate thedetection of LAR phosphatase activity in S2R� cells, weincreased the tyrosine phosphorylation of HA-tagged Liprin-�by coexpressing the Abelson (Abl) protein kinase (Fig. 2B),chosen because abl antagonizes Lar in the context of embryonicmotor axon guidance (35). Expression of the intracellulardomain of wild-type LAR decreased Liprin-� tyrosine phos-phorylation in the presence of Abl (Fig. 2B). As predicted,intracellular LAR constructs carrying the D1CS mutation aloneor in combination with the D1YL mutation did not reduceLiprin-� tyrosine phosphorylation (Fig. 2B). LAR-D1CS is thuscatalytically inactive on a functionally relevant substrate inDrosophila cells.

Phosphatase Activity Necessary for R7 Targeting Is Provided byPTP69D. The results above suggest that R7 targeting does notrequire LAR phosphatase activity. However, LAR is not the onlyRPTP involved in this process; loss of the other Drosophila typeIIa RPTP, PTP69D, from photoreceptors also causes R7 toterminate in the R8 layer (36)(Fig. S1A). Mutations that inac-tivate the remaining neuronally expressed RPTPs, PTP10D,PTP52F, and PTP99A, do not affect innervation of the R7 targetlayer (Fig. S2). To test whether R7 targeting requires thephosphatase activity of PTP69D, we used a UAS-PTP69Dtransgene with the catalytic aspartic acid in the WPD loop, whichis essential for phosphatase activity of PTP69D and othertyrosine phosphatases (7, 30), mutated to alanine (PTP69D-D1DA) (Fig. 1G) (37). PTP69D-D1DA had a significantly lowerability to rescue targeting of Ptp69D mutant R7 photoreceptorsthan wild-type PTP69D (Fig. S1E), indicating a requirement forPTP69D phosphatase activity.

We further found that removal of PTP69D created a require-ment for the phosphatase activity of LAR. Overexpression ofLAR in photoreceptors mutant for Ptp69D can rescue R7targeting (6). Although catalytically inactive LAR-D1CS res-cued R7 targeting to the same extent as wild-type LAR in Larmutants (Fig. S1E), wild-type LAR had a significantly greaterability to promote R7 targeting than LAR-D1CS in photore-ceptors singly mutant for Ptp69D or doubly mutant for bothPtp69D and Lar (Fig. S1 B–E). LAR is thus capable of dephos-phorylating substrates critical for R7 targeting, although inwild-type animals the necessary phosphatase activity is providedby PTP69D.

The Wedge Domain Is Required for LAR Dimerization and R7 Targeting.At the NMJ, a switch from activation of LAR by the ligand Sdcto inhibition of its phosphatase activity by Dlp is thought to allowmotor neurons to progress from synapse growth to active zonemorphogenesis (13). Since Sdc is present on photoreceptors (38),LAR is likely to be active in R7 unless downregulated by aninhibitory ligand. A constitutively active form of LAR wouldenable us to test the importance of LAR inhibition. Since forced

Fig. 1. Phosphatase activity of LAR is dispensable for R7 targeting. (A and B)Schematic representation of the photoreceptor axon projection pattern inwild-type (A) and Lar mutants (B). (C–E) Horizontal sections of adult headscarrying glass-lacZ to label all photoreceptors, stained with anti-�-galactosidase. Arrowheads indicate the target layers of R8 (blue) and R7 (red).(C) Wild-type; (D) Larc12/Lar2127; (E) Larc12/Lar2127, elav-GAL4; UAS-LARD1YLCS/�. (F) Horizontal sections like those shown in (C–E) were scoredfor R7 targeting defects. In Larc12/Lar2127, elav-GAL4 mutants, only 15% of R7axons project beyond the R8 layer, while addition of the indicated UAS-LARtransgenes significantly increased this percentage (FL 82%, D1CS 83%, D2CS81%, 2xCS 65%, D1YLCS 67%). A schematic of the LAR phosphatase domainswith the introduced point mutations in red is shown in (H). (G) Sequencecomparison of highly conserved structural motifs in domains D1 and D2 ofDrosophila LAR and PTP69D with the corresponding consensus sequences (32).Variable amino acid residues are shown in gray, residues deviating from theconsensus in blue, and residues mutated in this study are highlighted yellowin the consensus sequences and shown in red in the construct sequences.

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or ligand-induced dimerization inhibits the phosphatase activityof several RPTPs (16, 23, 39), we investigated the effect ofdimerization on LAR activity.

Two differently tagged LAR constructs, one of which lackedthe extracellular and transmembrane domains, and the other theD2 domain, could be co-immunoprecipitated from S2R� cells(Fig. 2 C and D), showing that intracellular domain interactionspromote LAR dimerization. Dimerization and inactivation ofRPTP� can be prevented by mutating two conserved prolineresidues in the juxtamembrane wedge domain (Fig. 2 A) (18, 21,23). We mutated the wedge proline residues (PP) in Drosophila

LAR to leucines (LL) to determine their effect on dimerization(Fig. 2A). Mutating the prolines in both monomers stronglyreduced their co-immunoprecipitation; however, the presence ofprolines in either monomer was sufficient for dimerization(Fig. 2C).

We next determined the effect of the wedge mutations onLAR catalytic activity. We found that the intracellular domainof LAR carrying the proline to leucine mutations (LAR-PPLL)was as active as wild-type LAR in reducing Abl-induced tyrosinephosphorylation of Liprin-� (Fig. 2B). We used NMJ synapsegrowth as an assay for phosphatase activity in vivo. A previousreport found that LAR-2XCS did not promote significant syn-apse growth in a Lar mutant background (13). We confirmed theimportance of LAR phosphatase activity at the NMJ usingUAS-LAR-D1CS, its non-substrate-trapping derivative UAS-LAR-D1YLCS, and UAS-LAR-2XCS. Neuronal expression ofthese constructs only slightly increased NMJ synapse growth ina Lar mutant background (Fig. 3 A, C, and E). The requirementfor LAR phosphatase activity at the NMJ is not due to absenceof PTP69D, since Ptp69D mutants also show reduced synapsesize (Fig. S3). Neuronally expressed UAS-LAR-PPLL promotedsynapse growth in Lar mutants to the same extent as wild-typeUAS-LAR (Fig. 3 B, D, and E), indicating that LAR-PPLL candephosphorylate the substrates relevant for NMJ growth.

Both assays indicate that LAR-PPLL is catalytically active; toaddress whether its activity is constitutive, we misexpressedLAR-PPLL and wild-type LAR in the wing imaginal disc, whichdoes not require Lar for its normal development. Ectopicexpression of LAR disrupted wing vein formation (Fig. S4),possibly by antagonizing the epidermal growth factor receptor,a receptor tyrosine kinase responsible for vein formation (40).Phosphatase activity is required for this function of LAR, since

Fig. 2. The wedge domain of LAR contributes to dimerization and R7targeting. (A) Sequence comparison of the Drosophila LAR and humanRPTP� wedge domains. Identical residues are shown in black, similar aminoacids in green and divergent residues in gray. Prolines 1,452/1,453 mutatedto leucine in LAR-PPLL are marked in red. (B) Wild-type LAR and LAR-PPLL,but not LAR-D1CS or LAR-D1YLCS, are catalytically active in cell culture.HA-tagged Liprin-� was immunoprecipitated from S2R� cells transfectedwith the indicated constructs and its tyrosine phosphorylation was as-sayed by Western blot (WB) with anti-phosphotyrosine (pY), normalizedto anti-HA in the immunoprecipitate. Co-transfection of Abl increasedLiprin-� pY levels (compare lane 5 to 6). In the presence of Abl (lanes 1–5),co-transfection of the intracellular domain of wild-type LAR (1) or LAR-PPLL(4) reduced pY levels on Liprin-�. Co-transfection of the intracellulardomain of LAR-D1CS (2) or LAR-D1YLCS (3) did not reduce Liprin-� pYlevels. (C) Co-immunoprecipitation of the Myc-tagged intracellular domainof LAR and HA-tagged LAR lacking the D2 domain expressed in S2R� cells.Wild-type (lanes 1 and 3) or PPLL mutant Myc-LAR-intra (lanes 2 and 4) waspulled down from cell lysates using anti-Myc antibody. Levels of co-immunoprecipitated wild-type (lanes 1 and 2) or PPLL mutant (lanes 3 and4) HA-LAR�D2 were assayed by WB (top row). After normalization to bothimmunoprecipitated Myc-LAR-intra (2nd row) and HA-LAR�D2 in the input(third row), there was a negligible difference in wild-type HA-LAR�D2co-immunoprecipitated with PPLL mutant Myc-LAR-intra (2) compared towild-type Myc-LAR-intra (1). There was a 5-fold decrease in PPLL mutantHA-LAR�D2 co-immunoprecipitated with PPLL mutant Myc-LAR-intra (4)compared to wild-type Myc-LAR-intra (3). (D) Schematic of a putative dimerformed by the tagged LAR constructs used in (C), indicating the wedgedomain (W). (E) Quantification of R7 targeting defects in horizontal sec-tions of adult heads. While elav-GAL4-driven expression of wild-type LAR(82%) or LAR-D1CS (83%) strongly rescues R7 targeting in Lar null mutants(15%), wedge mutant LAR-PPLL has a reduced ability to promote R7targeting (40%), which is not due to hyperactivity since catalytically inac-tive LAR-PPLL-D1CS shows a similarly low percentage of correctly targetedR7 axons (49%). Double asterisks indicate statistical significance (P � 0.0001).

Fig. 3. NMJ growth requires LAR catalytic activity, but not an intact wedgedomain. (A–D) Neuromuscular junctions at muscle 6/7 in segment A2 offilleted third-instar larvae were stained with anti-HRP (magenta) to label allneuronal membranes and anti-Syt (green) to label synaptic boutons. (A)Larc12/Lar2127, elav-GAL4; (B) Larc12/Lar2127, elav-GAL4; UAS-LAR/�; (C) Larc12/Lar2127, elav-GAL4; UAS-LAR-D1CS/�; (D) Larc12/Lar2127, elav-GAL4; UAS-LARPPLL/�. (E) Quantification of bouton number in segment A2 at the muscle6/7 synapse. Compared to the number of boutons in Lar null mutants (63 � 23),wild-type LAR (148 � 25) and wedge mutant LAR-PPLL (143 � 36) promoteequivalent levels of synapse growth. Catalytically inactive forms of LAR showless ability to promote synapse growth (LAR-D1CS: 108 � 29, LAR-D1YLCS:102 � 26, LAR-2XCS: 94 � 17). Statistically significant differences are indicatedby ** (P � 0.0001) or * (P � 0.005).

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expression of LAR-2XCS did not cause vein defects (Fig. S4).Ectopic expression of LAR-PPLL caused a greater frequencyand severity of vein formation defects than wild-type LAR (Fig.S4), suggesting an increase in its phosphatase activity.

We next determined the effect of LAR-PPLL on R7 targeting.In a wild-type background, expression of LAR-PPLL in R7 hadno apparent effect (Fig. S5), perhaps due to its dimerization withand inhibition by endogenous LAR. However, UAS-LAR-PPLLhad much less ability to rescue R7 targeting in Lar mutants thanwild-type UAS-LAR (Fig. 2E), although it was correctly local-ized to photoreceptor growth cones (Fig. S6). If this R7 targetingdefect reflected a detrimental effect of constitutive phosphataseactivity, then inactivating the phosphatase domain of LAR-PPLL by introducing the D1CS mutation should restore its R7targeting function. However, in a Lar mutant background UAS-LAR-PPLL-D1CS showed weak R7 rescuing activity similar toUAS-LAR-PPLL, rather than strong rescuing activity like UAS-LAR-D1CS (Fig. 2E). Since eliminating its phosphatase activitydoes not restore the function of LAR-PPLL, it is unlikely that therole of the wedge prolines is to allow inhibition of LARphosphatase activity. Taken together, these findings show thatLAR phosphatase activity neither promotes nor inhibits R7targeting, and are consistent with a non-catalytic role for LARin this process.

LAR Function in R7 May Be Controlled by an Uncharacterized Ligand.The HSPGs Sdc and Dlp are thought to regulate LAR phos-phatase activity in NMJ morphogenesis and motor axon guid-ance (12, 13). It is unclear whether the same ligands control thephosphatase-independent function of LAR in R7 targeting,since sdc and dlp mutations have complex effects on photore-ceptor projection patterns (38). Sdc and Dlp bind exclusively tothe three Ig domains of LAR (12, 13). Since a UAS-LARtransgene lacking the three Ig domains (8) strongly rescued R7targeting in a Lar mutant background (Fig. 4 A and B), Sdc andDlp are unlikely to be the relevant ligands for this process.

In contrast, deletion of 8 of the 9 FNIII domains(LAR�FNIII2–9) (8) strongly reduced the ability of LAR torescue R7 targeting (Fig. 4 A and B). We used deletion con-structs lacking subsets of FNIII domains to analyze whetherindividual FNIII domains differ in their ability to promote R7targeting. R7 targeting was rescued by neuronal expression ofconstructs lacking either FNIII domains 1–3 or FNIII domains4–6 (Fig. 4 A and B). Only loss of the three most proximal FNIIIdomains, 7–9, caused a decrease in rescuing activity similar todeletion of domains 2–9 (Fig. 4 A–C). To test whether thesedomains were not only necessary, but also sufficient, to promoteR7 targeting, we generated a UAS-LAR transgene with anextracellular domain solely composed of FNIII domains 7–9(LAR�Ig�FNIII1–6). Neuronal expression of this constructstrongly rescued R7 targeting (Fig. 4 A, B, and D). FNIIIdomains 7–9 are therefore sufficient to promote R7 targeting,and are likely to bind a novel ligand that regulates the non-catalytic function of LAR. The function of LAR in R7 thusdiffers from its function at the NMJ in three respects: itsindependence of intrinsic phosphatase activity, its requirementfor an intact wedge domain, and its regulation by a distinctextracellular ligand.

DiscussionWe have shown that a catalytically inactive form of LAR(LAR-D1CS) can promote R7 targeting. Its function in R7 isnot due to substrate trapping, since it does not require thetyrosine residue that stabilizes substrate binding in the activesite (31). Conversely, a mutant form of LAR that retainsphosphatase activity in vitro and phosphatase-dependent func-tions in vivo (LAR-PPLL) fails to promote R7 targeting. Thisdefect is not due to unregulated LAR phosphatase activity,

because mutating the active site of LAR-PPLL does notrestore its function in R7. We therefore conclude that R7targeting is independent of LAR catalytic activity. In contrast,the phosphatase activity of the RPTP PTP69D is required forthis process. Our rescue experiments suggest that LAR hasretained the ability to dephosphorylate substrates of PTP69D,perhaps because it acts on the same molecules in otherprocesses such as NMJ synapse growth. PTP69D overexpres-sion does not rescue R7 targeting in Lar mutants (6), suggest-ing that LAR has evolved a second function.

Several elements of the LAR protein are essential for its non-catalytic function in R7. Within the extracellular domain, FNIIIdomains 7–9 are necessary and sufficient to promote R7 targeting.The identified ligands for Drosophila and vertebrate LAR do notinteract with these domains, suggesting the involvement of anunknown ligand (Fig. 4E). In Lar mutants, R7 axons initially project

Fig. 4. R7 targeting requires only FNIII domains 7–9 in the LAR extracel-lular domain. (A) A schematic of the different LAR extracellular deletionconstructs. Squares indicate FNIII domains and circles Ig domains. (B)Quantification of R7 targeting defects. In Larc12/Lar2127, elav-GAL4 mutantsonly 20% of R7 axons are correctly targeted. Pan-neuronal expression ofthe UAS-LAR extracellular deletion constructs rescues R7 targeting if theyretain FNIII domains 789 (�Ig123 73%, �FNIII123 69%, �FNIII456 81%,�Ig�FNIII1– 6 75%, FL 83%). Constructs lacking FNIII789 do not rescue R7targeting (�FNIII2–9 10%, �FNIII789 13%). (C and D) Horizontal sections ofadult heads carrying glass-lacZ and stained with anti-�-galactosidase. Ar-rowheads indicate the terminal layers of R8 (blue) and R7 (red). (C) Larc12/Lar2127, elav-GAL4; UAS-LAR�FNIII789/�. (D) Larc12/Lar2127, elav-GAL4; UAS-LAR�Ig�FNIII1– 6/�. (E) Distinct mechanisms of LAR signaling in Drosophilaneurons. LAR (purple) may promote synapse growth as a catalytically activemonomer with an unpaired wedge domain (W) that can dephosphorylatetyrosine-phosphorylated substrates (pY, red). The HSPG ligand Sdc acts inmotor neurons to increase LAR activity by binding to its Ig domains (circle).Active zone morphogenesis requires competitive binding of the inhibitoryHSPG ligand Dlp expressed in muscle cells to the Ig domains. Dlp inhibitsLAR phosphatase activity, perhaps by inducing dimerization through in-sertion of the wedge domain of one monomer into the active site of theother. R7 targeting requires binding of an unknown ligand (L) to FNIIIdomains 7–9 (square), and an intact wedge domain. Dimerization medi-ated by the wedge domain may result in conformational changes thatallow binding of a transmembrane ligand presented by the medulla targetcells, or an intracellular adaptor protein (green). This scaffolding functionof LAR might increase adhesion to target neurons through interactionswith an intracellular protein network (yellow).

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beyond R8, but then retract back to the R8 layer (4, 6), suggestingthat LAR stabilizes adhesion between R7 and its target cells.Interaction with a transmembrane rather than a secreted ligandwould best direct the formation of stable contacts with specificmedulla target neurons. Interestingly, the transmembrane proteinNetrin-G ligand-3 has recently been shown to interact with verte-brate LAR, although its binding site on LAR is unknown (41).While interactions of the Ig domains with the HSPGs Sdc andDlp regulate the catalytic activity of LAR, ligand binding to theFNIII domains might induce conformational changes that revealbinding sites for intracellular adaptor proteins, or control proteo-lytic cleavage, which regulates the association of vertebrate LARwith �-catenin (42). Ligand binding might also localize LAR to aspecific region in the R7 growth cone.

In the intracellular domain, the two juxtamembrane prolineresidues required for R7 targeting are homologous to prolinesthat stabilize the wedge domain of RPTP�, which mediatescatalytic inhibition through dimerization (18, 21, 23). AlthoughLAR function in R7 is phosphatase-independent, it may requiredimerization (Fig. 4E). Our co-immunoprecipitation assays sup-port the existence of LAR homodimers in Drosophila cells,consistent with reports of RPTP dimers in vertebrate cell types(17–20). However, structural studies of LAR family RPTPssuggest that steric hindrance by the D2 domains would preventthe wedge-mediated mode of dimerization (33, 43). Mutations inthe wedge prolines may affect LAR dimerization because therelative orientations of D1 and D2 are flexible in vivo, or becausethe wedge contributes to dimerization mediated by interactionsbetween D1 and D2 domains (20, 43, 44), or their effect may beindirect. Consistent with an essential role for dimerization, apeptide homologous to the wedge specifically inhibits the bio-logical functions of LAR in vertebrate cells (45). Interestingly, awedge mutation in the mouse RPTP CD45 has opposite effectson T-cell response in central and peripheral T cells (46). Thissuggests that RPTPs other than LAR may also signal in bothcatalytic and non-catalytic modes that are affected differently bychanges in the wedge domain.

Dimerization could affect the ability of LAR to bind eitherdownstream signal transduction molecules or upstream ligands(17). Ligand-regulated binding of adaptor proteins, such asLiprin-�, might assemble a protein network that mediates func-tions independent of LAR phosphatase activity (Fig. 4E). Mostintracellular binding partners of LAR, including Abl, Enabledand Liprin-�, interact with the catalytically inactive D2 domain(3, 10, 35), which is essential for R7 targeting (10). In vertebrates,large PTPs that contain protein interaction domains such asFERM or PDZ domains can act independently of phosphataseactivity (47). Perhaps the catalytically inactive PTP-D2 domainsof LAR and other RPTPs have evolved to act as protein dockingsites.

We show here that LAR uses two very different modes ofsignaling at the NMJ and in R7. R7 targeting employs a liganddistinct from the HSPGs Sdc and Dlp that control LAR activityat the NMJ (13). While the catalytic activity of LAR is requiredto promote NMJ synapse growth (13), it is dispensable for R7targeting. Inhibition of LAR catalytic activity also plays no rolein R7 targeting, although it is essential for active zone morpho-genesis (13). Finally, an intact wedge domain is necessary forLAR to function in R7, but not in NMJ growth. Synapse growthand R7 targeting are likely to involve different cellular responsesto LAR signaling. The R7 targeting defect reflects reducedadhesion to target neurons (4, 6), while NMJ growth is achievedby sprouting and extension of new axonal branches. The twosignaling modes probably require distinct downstream factors, inaddition to common components such as Liprin-� (3, 6, 10, 34,48). The different requirements for LAR structural motifs makeit possible to selectively interfere with only a subset of LAR-dependent processes. Since LAR and other RPTPs are impli-

cated in various human diseases (49–51), differential usage ofsignaling modes may prove important for the development ofselective pharmaceutical reagents.

Materials and MethodsGenetics. UAS-HA-LAR transgenic flies were made with the construct de-scribed in (10). UAS-LAR extracellular deletion constructs (�Ig123, �FNIII123,�FNIII456, �FNIII789 and �FNIII2–9) and active site mutations (D1CS, D2CS, and2xCS) were described in (8). Other stocks used were Lar2127, LarC12 and UAS-LAR(6), glass-lacZ (52), elav-GAL4, and sev-GAL4 (Bloomington Drosophila StockCenter).

Transgenes and Expression Constructs. To make UAS-HA-LAR-�Ig123�FNIII1–6, the signal sequence from Wingless followed by three copiesof the HA epitope tag (36) was fused in frame to the N terminus of FNIII domain7 of LAR. This construct contains LAR amino acids 907–2,029. UAS-HA-LARcontains LAR amino acids 33–2,029 (10). UAS-HA-LAR-�D2 was derived fromthis construct using PCR to delete amino acids 1,811–2,024 of LAR. pPAC-Myc-LARintra was made by adding five copies of the Myc epitope tag to the Nterminus of the LAR intracellular domain (LAR amino acids 1,415–2,029) in thepPAC-PL vector. Mutagenic primers were used to introduce the point muta-tions D1-C1670S, D1-Y1504L and PP1452/1453LL into the corresponding LARconstructs by PCR. The Abl-Myc sequence inserted into pPAC-PL to generatepPAC-Abl-Myc was from pMET-Abl-Myc (53).

Immunohistochemistry. Primary antibodies used were rabbit anti-�-galactosidase (1:2,500; Cappel) and rabbit anti-Synaptotagmin (Syt; 1:2,000; agift from H. Bellen). Primary antibody incubations were performed overnightat 4 °C. Fluorescently conjugated secondary antibodies and goat anti-horseradish peroxidase (HRP; Jackson ImmunoResearch) were used at 1:250for 2 h at room temperature. Fluorescent images were collected on a ZeissLSM510 confocal microscope. Adult head sections were stained as described(36). Larval NMJs were stained as described (3).

Quantification of Phenotypic Defects. R7 targeting was quantified as previ-ously described (10). Larval fillets stained with anti-HRP and anti-Syt wereused to obtain maximum intensity projections of stacks of confocal imagesof the muscle 6/7 synapse in abdominal segment 2. Boutons per synapsewere counted blind in groups of 2–5 genotypes. Statistical analysis of allquantifications used Microsoft Excel and Student’s t-test (http://www.physics.csbsju.edu/stats/t test.html). Error bars indicate standard de-viations. One transgenic line per construct was tested with the followingexceptions: data from more than one line were pooled for LAR-YLD1CS(2�), LAR-PPLL (2�), LAR-PPLLD1CS (3�), LAR-�Ig123�FNIII1– 6 (3�), andLAR-WT (2� only in Fig. 3).

Co-Immunoprecipitation (Co-IP) and Phospho-Tyrosine (pY) Detection. Cultur-ing and transfection of S2R� cells was performed as described (10). Forty-eight hours after transient transfection, S2R� cells were rinsed briefly in PBS(coIP) or ddH2O (pY) and lysed in ice-cold IP buffer [50 mM Tris, pH 7.5, 150 mMNaCl, 1 mM NaF, and 1% Nonidet P-40 with protease inhibitors (Roche)]including 1 mM EDTA for pY detection. Proteins were immunoprecipitatedwith 2 �g mouse anti-Myc 9E10 (Santa Cruz Biotechnology) or 1.5 �g ratanti-HA 3F10 (Roche). Protein-G agarose beads (Roche) were used for precip-itation according to the manufacturer’s instructions. Proteins were separatedby SDS/PAGE and probed with mouse anti-Myc 9E10 (1:8,000), rat anti-HA 3F10(1:1,000), or mouse anti p-Tyr (PY99)-HRP (Santa Cruz Biotechnology; 1:500) inTris-buffered saline/0.2% Tween 20 with 5% BSA, followed by HRP-coupleddonkey anti-mouse or anti-rat (1:8,000; Jackson ImmunoResearch). Signal wasdetected by enhanced chemiluminescence (Pierce). For signal quantification,scanned image files were analyzed using the Photoshop histogram tool tomeasure band intensity.

Additional methods are described in SI Text.

ACKNOWLEDGMENTS. We thank Hugo Bellen (Baylor College of Medicine),Catherine Collins (University of Michigan), Barry Dickson (Institute of Molec-ular Pathology, Vienna), David Forsthoefel (Ohio State University), Paul Gar-rity (Brandeis University), Mark Seeger (Ohio State University), Helen Sink(NYU School of Medicine), David Van Vactor (Harvard Medical School), theBloomington Drosophila stock center, and the Developmental Studies Hybrid-oma Bank for fly stocks, reagents and technical advice and Sergio Astigarraga,Katrin Deininger, Claude Desplan, Thomas Hurd, Kevin Legent, Sylvie OzonRickman, Jean-Yves Roignant, and Josie Steinhauer for critical comments onthe manuscript. This work was supported by March of Dimes Birth DefectsFoundation Grant 1-FY04-101.

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