Author response: Context-dependent requirement of G protein coupling for Latrophilin-2 in target selection of hippocampal axons

Abstract

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The formation of neural circuits requires extensive interactions of cell-surface proteins to guide axons to their correct target neurons. Trans-cellular interactions of the adhesion G protein-coupled receptor latrophilin-2 (Lphn2) with its partner teneurin-3 instruct the precise assembly of hippocampal networks by reciprocal repulsion. Lphn2 acts as a repulsive receptor in distal CA1 neurons to direct their axons to the proximal subiculum, and as a repulsive ligand in the proximal subiculum to direct proximal CA1 axons to the distal subiculum. It remains unclear if Lphn2-mediated intracellular signaling is required for its role in either context. Here, we show that Lphn2 couples to Gα12/13 in heterologous cells; this coupling is increased by constitutive exposure of the tethered agonist. Specific mutations of Lphn2’s tethered agonist region disrupt its G protein coupling and autoproteolytic cleavage, whereas mutating the autoproteolytic cleavage site alone prevents cleavage but preserves a functional tethered agonist. Using an in vivo misexpression assay, we demonstrate that wild-type Lphn2 misdirects proximal CA1 axons to the proximal subiculum and that Lphn2 tethered agonist activity is required for its role as a repulsive receptor in axons. By contrast, neither tethered agonist activity nor autoproteolysis were necessary for Lphn2’s role as a repulsive ligand in the subiculum target neurons. Thus, tethered agonist activity is required for Lphn2-mediated neural circuit assembly in a context-dependent manner. Editor's evaluation This is an intriguing study investigating the molecular mechanisms of neural circuit developmental organization. Using a defined hippocampal circuit, the authors find that ectopic expression of an adhesion G protein receptor leads to axon mistargeting. This work defines new mechanisms of axon target specificity. https://doi.org/10.7554/eLife.83529.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest The complex brain circuits that allow animals to sense and interact with their environment start to form early during development. Throughout this period, neurons extend fiber-like projections to establish precise wiring patterns. Various types of proteins at the surface of both incoming fibers and target cells ensure that only the right partners will connect together. Latrophilin-2, for example, is a neuronal surface protein essential for the formation of accurate connections in the hippocampus, a brain region important for memory. Studded through the membrane of certain neurons, it acts as a signal-sending ligand to direct incoming fibers, with neurons that carry Latrophilin-2 repelling projections from cells that display certain protein partners. At the same time, Latrophilin-2 also allows neurons to receive chemical signals by working with intracellular signaling proteins known as G proteins, which help to relay information between cells. It remained unclear how this role as a signalling receptor participates in the wiring of the hippocampus during development. To explore this question, Pederick, Perry-Hauser et al. examined the impact of Latrophilin-2 on the connection patterns of mouse hippocampal neurons that do not normally carry this protein. Introducing Latrophilin-2 into these ‘proximal CA1 cells’ misdirected them away from their usual partners – unless Latrophilin-2 was altered so that it could not interact with G proteins. In contrast, forcing the connecting partners of CA1 cells to display normal or altered versions of Latrophilin-2 did not interfere with the protein acting as a repulsive ligand. Taken together, these results suggest that the ability of Latrophilin-2 to signal through G proteins is important for neurons that are attempting to project their fibers onto other cells, but not important when Latrophilin-2 acts in targets to direct incoming fibers from other neurons. These results show that a single protein can shape neural circuits by acting both as a signal-receiving receptor and a signal-sending ligand depending on the context. In the future, Pederick, Perry-Hauser et al. hope that their findings will shed new light on how the wiring of the brain is disrupted in neurodevelopmental disorders. Introduction Latrophilins (Lphn1–3) are highly expressed in the brain and were originally identified as responders to ɑ-latrotoxin, a neurotoxin from black widow spider venom that causes the profound release of neurotransmitters from nerve terminals (Davletov et al., 1996). They belong to the family of adhesion G protein-coupled receptors (aGPCRs), capable of eliciting intracellular effects through coupling with heterotrimeric G proteins (Lelianova et al., 1997). Additionally, as cell adhesion molecules, latrophilins interact via their N-terminal extracellular domain with four different families of interacting partners including neurexins (Boucard et al., 2012), teneurins (Silva et al., 2011), fibronectin leucine-rich transmembrane proteins (FLRTs) (O’Sullivan et al., 2012), and contactins (Zuko et al., 2016). In the central nervous system, latrophilins have been implicated in neuronal migration, circuit assembly, and synapse formation (Anderson et al., 2017; Del Toro et al., 2020; Donohue et al., 2021; Pederick et al., 2021; Sando et al., 2019; Sando and Südhof, 2021). In humans, polymorphisms in LPHN3 are associated with an increased risk of attention-deficit/hyperactivity disorder, and a missense variant in the LPHN2 gene is responsible for extreme microcephaly (Arcos-Burgos et al., 2010; Domené et al., 2011; Vezain et al., 2018). We recently showed in mice that one of the three latrophilins, Lphn2, displays expression patterns inverse to teneurin-3 (Ten3) in two parallel hippocampal networks (Pederick et al., 2021). While hippocampal Lphn2 is preferentially expressed in the distal CA1 and the proximal subiculum, Ten3 is enriched in the proximal CA1 and the distal subiculum. These expression patterns and reciprocal repulsions mediated by Ten3-Lphn2 interactions instruct proximal CA1 axons to target the distal subiculum, and more distal CA1 axons to target more proximal subiculum (Figure 1A). Specifically, Lphn2 acts as a ‘receptor’ in more distal CA1 axons that is repelled by Ten3 expressed from the distal subiculum (Figure 1B). At the same time, Lphn2 acts as a repulsive ‘ligand’ in the proximal subiculum to repel Ten3-expressing (Ten3+) proximal CA1 axons; this action requires Lphn2’s teneurin-binding domain but not its FLRT-binding activity (Figure 1C; Pederick et al., 2021). Therefore, Lphn2 is required cell autonomously as a receptor in more distal CA1 axons for their precise target selection, and non-autonomously in target neurons as a ligand for precise target selection of proximal CA1 axons. While Ten3 additionally mediates homophilic attraction (Berns et al., 2018; Pederick et al., 2021), Lphn2 does not mediate homophilic binding in trans (Boucard et al., 2014; Pederick et al., 2021). Figure 1 with 3 supplements see all Download asset Open asset Misexpression of latrophilin-2 (Lphn2) in proximal CA1 axons causes axon mistargeting to the proximal subiculum. (A) Cartoon depicting the topographic connections from proximal CA1 (pCA1) to distal subiculum (dSub) and distal CA1 (dCA1) to proximal subiculum (pSub). Ten3+ proximal CA1 axons are repelled from Lphn2 expressing (Lphn2+) proximal subiculum and Lphn2+ axons are repelled from Ten3+ distal subiculum. Red symbols indicate the repulsive cues experienced by CA1 axons, previously described in Pederick et al., 2021. (B) Deletion of Lphn2 from CA1 leads to distal CA1 axons mistargeting to distal subiculum, suggesting that Lphn2 acts cell-autonomously as a repulsive receptor. (C) Deletion of Lphn2 from proximal subiculum results in proximal CA1 axon mistargeting to proximal subiculum, suggesting Lphn2 acts cell-non-autonomously as a repulsive ligand. Figures (A–C) are based on Pederick et al., 2021. (D) Experimental design of Lphn2 misexpression assay in proximal CA1. At postnatal day (P) 0, lentivirus expressing Cre or Cre and Lphn2 was injected into CA1. This was followed by injection at P42 of Cre-dependent membrane bound mCherry (mCh) into proximal CA1 as an axon tracer. (E and F) Representative images of AAV-DIO-mCh (magenta; mCh expression in a Cre-dependent manner) injections in proximal CA1 (top) and corresponding projections in the subiculum (bottom). (G) A representative image of the subiculum with proximal subiculum (pSub), mid subiculum (mSub), and distal subiculum (dSub) regions highlighted. (H) The fraction of total axon intensity within proximal, mid, and distal subiculum. Cre: N=5 and Cre-Lphn2: N=5. Means ± SEM; two-way ANOVA with Sidak’s multiple comparisons test. Injection sites of all subjects are shown in Figure 1—figure supplement 3. Scale bars represent 200 μm. Figure 1—source data 1 Misexpression of latrophilin-2 (Lphn2) in proximal CA1 axons causes axon mistargeting to the proximal subiculum. https://cdn.elifesciences.org/articles/83529/elife-83529-fig1-data1-v2.zip Download elife-83529-fig1-data1-v2.zip Structurally, the N-terminal extracellular domain of latrophilins comprises a rhamnose-binding lectin (RBL) domain, an olfactomedin-like (OLF) ligand-binding domain, a serine/threonine-rich region and hormone receptor motif (HRM), and a conserved GPCR autoproteolysis-inducing (GAIN) domain that encompasses the GPCR proteolysis site (GPS) (Araç et al., 2012; Moreno-Salinas et al., 2019; Vizurraga et al., 2020; Figure 2A). aGPCRs undergo autoproteolytic cleavage at the HL/T consensus site within the GPS. This self-cleavage divides the receptor into an extracellular N-terminal fragment (NTF) and a membrane-bound C-terminal fragment (CTF) that remain noncovalently associated throughout biosynthesis and membrane trafficking (Vizurraga et al., 2020). The seven residues immediately C-terminal to the GPS constitute the tethered agonist peptide (also known as the Stachel or stalk peptide), which upon exposure binds within the transmembrane domain to activate heterotrimeric G proteins (Liebscher and Schöneberg, 2016). Figure 2 with 2 supplements see all Download asset Open asset Exposure of the latrophilin-2 (Lphn2) tethered agonist (TA) promotes intracellular signaling through Gα12/13. (A) Cartoon representations of full-length and tethered agonist-exposed (CTF) Lphn2 with detailed amino acid sequences for the TA. The extracellular domain of Lphn2 comprises an N-terminal rhamnose-binding lectin domain (RBL), an olfactomedin-like domain (OLF), a serine/threonine-rich region, and a HormR domain (HRM). It also contains the GPCR autoproteolysis-inducing (GAIN) domain necessary for autoproteolytic cleavage. This cleavage divides the aGPCR into two polypeptide chains: an N-terminal fragment (NTF) and a C-terminal fragment (CTF). The peptide stretch directly following the proteolytic cleavage site is known as the ‘Stachel’ or tethered agonist. Exposure of the tethered agonist results in aGPCR activation and downstream signaling. (B) Representative immunoblot analysis (N=3) of wild-type Lphn2 and Lphn2-CTF expression in HEK293T cells using a primary antibody against FLAG (1:500, ThermoFisher, PA1-984B). Expected bands for full-length Lphn2-FLAG and Lphn2-CTF-FLAG are 164 kDa and 72 kDa, respectively. (C) Serum response element (SRE) luciferase reporter assay for Lphn2 and Lphn2-CTF shows that removing the entire NTF up to the GPS cleavage site constitutively enhances SRE signaling (N=3 biological replicates, 9 technical replicates). (D) Schematic outlining the Gβγ-release bioluminescence resonance energy transfer (BRET) assay. The Lphn2 tethered agonist is capped with an enterokinase cleavage site (EK) preceded by a hemagglutinin signal peptide (SP), the P2Y12 N-terminal extracellular sequence, and a flexible linker (Lizano et al., 2021). Addition of 10 nM enterokinase generates a tethered agonist neoepitope identical to activated endogenous Lphn2. Lphn2 activation results in G protein dissociation, allowing Gβγ-Venus to associate with the C-terminus of GPCR kinase 3 (GRK3-ct) (Hollins et al., 2009). (E) Gβγ-release BRET assay testing SP-P2Y12-EK-Lphn2-CTF activation of Gαs, Gαi, Gαq, Gα12, and Gα13 in HEKΔ7 cells (N=3–4 biological replicates, 9–12 technical replicates). β2-adrenergic receptor (β2AR) with 1 µM isoproteronal, dopamine receptor D2 (D2R) with 10 µM quinpirole, and endothelin receptor (ETA) with 100 nM ligand ET-1 were used as positive controls, for Gαs, Gαi and Gαq/12/13 ,respectively. Means ± SEM; Multiple unpaired t tests between no G protein and G protein conditions; **p<0.01; ****p<0.0001. Figure 2—source data 1 Full raw unedited blot of latrophilin-2 (Lphn2) expression in HEK293T cells. Primary antibody against Flag (1:500, ThermoFisher, PA1-984B), secondary antibody anti-rabbit HRP (1:10,000, ThermoFisher, Cat #31458). The same blot was used in Figure 3B and Figure 4A. https://cdn.elifesciences.org/articles/83529/elife-83529-fig2-data1-v2.zip Download elife-83529-fig2-data1-v2.zip Figure 2—source data 2 Uncropped immunoblot analysis of latrophilin-2 (Lphn2) expression in HEK293T cells. Primary antibody against Flag (1:500, ThermoFisher, PA1-984B), secondary antibody anti-rabbit HRP (1:10,000, ThermoFisher, Cat #31458). Blue arrows indicate bands of interest for Lphn2-CTF-Flag and full-length Lphn2-Flag. https://cdn.elifesciences.org/articles/83529/elife-83529-fig2-data2-v2.zip Download elife-83529-fig2-data2-v2.zip Figure 2—source data 3 Replicates of the immunoblot assay. Replicate four was used for Figures 2—4. https://cdn.elifesciences.org/articles/83529/elife-83529-fig2-data3-v2.zip Download elife-83529-fig2-data3-v2.zip While our previous in vivo work established that interaction between Ten3 and Lphn2 was required for precise circuit assembly (Pederick et al., 2021), it did not examine how this might depend on Lphn2-mediated signaling mechanisms. Here, we modified our previous hippocampal model to develop an Lphn2 misexpression assay (Figure 1D). We misexpressed Lphn2 in either CA1 axons or the subiculum target and assessed the impact on normal proximal CA1→distal subiculum axon targeting. We found that ectopically expressing wild-type Lphn2 in proximal CA1 axons causes their mistargeting to the proximal subiculum. This provided us with a robust platform to interrogate whether tethered agonist activity or autoproteolytic cleavage is required for axon mistargeting in this Lphn2 ectopic expression system. When misexpressed in CA1, Lphn2 tethered-agonist activity was required for Lphn2-mediated axon mistargeting. By contrast, when we misexpressed Lphn2 in subiculum target neurons, both tethered agonist activity and autoproteolysis were dispensable for Lphn2-mediated axon repulsion. Thus, our data support that Lphn2 G-protein coupling is required in axons but not target neurons during precise circuit assembly. Results Misexpression of wild-type Lphn2 in proximal CA1 leads to axon mistargeting in the subiculum To investigate the role of Lphn2-mediated G protein activity in hippocampal axon targeting, we first designed a gain-of-function assay in which we misexpressed Lphn2 in proximal CA1 neurons. We hypothesized that this ectopic expression would cause proximal CA1 axons to avoid the Ten3+ distal subiculum and incorrectly target the proximal subiculum. If so, this platform could provide us with an assay to test Lphn2 mutants with defects in various functions to determine whether wild-type Lphn2 mistargeting is compromised. To test our hypothesis, we used a dual injection strategy to ectopically express Lphn2 in proximal CA1 and trace its axons into the subiculum (Figure 1—figure supplement 1). At postnatal day 0 (P0), lentivirus expressing Cre (LV-Cre) (control) or Cre-Lphn2 (LV-Cre-P2A-Lphn2) was injected into proximal CA1, followed by injection of a Cre-dependent membrane-bound mCherry (AAV-DIO-mCherry) into proximal CA1 in the same mice at approximately P42 (Figure 1D). We confirmed the expression of Lphn2 in CA1 axons by the presence of FLAG immunostaining in the subiculum and that ectopic expression levels were higher than that of endogenous Lphn2 (Figure 1—figure supplement 2A, C and D). As expected, in control animals (Cre), Cre expressing (Cre+) proximal CA1 axons targeted the most distal parts of the subiculum (Figure 1E). By contrast, when Lphn2 was misexpressed in proximal CA1 (Cre-Lphn2), Cre+ proximal CA1 axons targeted the most proximal parts of the subiculum (Figure 1F). To analyze the location of proximal CA1 axons in the subiculum, we calculated the fraction of axon intensity within thirds of the subiculum across the proximal/distal axis (Figure 1G). Proximal CA1 axons misexpressing Lphn2 are located significantly more in the proximal third of the subiculum and significantly less in the distal third of the subiculum when compared to control axons (Figure 1H). These data supported our hypothesis that ectopic expression of Lphn2 in proximal CA1 axons causes mistargeting to the proximal subiculum. Importantly, the phenotype observed when overexpressing Lphn2 in pCA1 axons is more severe than that observed when Ten3 is deleted (Berns et al., 2018), suggesting that mistargeting is not caused by disruption of Ten3 expression alone. Having established the effect of wild-type Lphn2 misexpression in proximal CA1 axons, we next sought to characterize G protein coupling of wild-type Lphn2 and generate Lphn2 mutants to test the requirement of G protein signaling in Lphn2 mediated neural circuit assembly. Lphn2 signals through Gα12/13 The G protein interaction partners for Lphn2 have not been previously established. We recently showed that Lphn3, another member of the latrophilin family of aGPCRs, couples principally to Gα12/13, and also more weakly to Gαq, using a combination of gene expression assays and an activation strategy that permitted acute exposure of the tethered agonist in a live-cell system (Mathiasen et al., 2020). Thus, we began our signaling characterization of Lphn2 similarly using a wild-type full-length Lphn2 construct, and a constitutively active construct termed Lphn2-CTF (Figure 2A). The wild-type Lphn2 construct comprises all extracellular elements including the RBL, OLF, HRM, and GAIN domains, in addition to the seven transmembrane helix domain. The Lphn2-CTF lacks the entire NTF up to the GPS and instead has only a methionine residue before the tethered agonist. We tested the expression of these constructs in mammalian cells using immunoblotting and showed that both full-length Lphn2 and Lphn2-CTF ran at the expected truncated position (~72 kDa) suggesting that full-length Lphn2 undergoes normal proteolytic cleavage (Figure 2B). This result for full-length Lphn2 is similar to our work characterizing autoproteolysis of Lphn3 (Perry-Hauser et al., 2022). To infer the activity of these constructs in G protein signaling pathways, we used a luminescence-based gene expression assay for serum response element (SRE), which produced a robust response in our previous studies of Lphn3 (Mathiasen et al., 2020). In our assay design, SRE action is coupled to the transcription and translation of firefly luciferase; this readout is then normalized to the control reporter, Renilla luciferase, expressed from the same plasmid under a constitutive promoter. We found that Lphn2-CTF significantly enhanced signaling over wild-type Lphn2 for SRE gene expression at varying levels of cDNA transfection (Figure 2C). Since the SRE assay reports on signaling by Gα12/13 as well as Gαq we tested whether Gα12/13 or Gαq was the primary contributor to this response using a selective Gαq inhibitor, YM-254890 (Figure 2—figure supplement 1). We did not observe a significant effect upon the addition of the inhibitor, suggesting that Lphn2 signals through Gα12/13. To verify our result in the context of acute G protein activation, we next tested how tethered agonist exposure affects G protein activation in a bioluminescence resonance energy transfer (BRET) assay (Figure 2D). We designed a synthetically-activatable Lphn2 construct based on a recent publication that took advantage of the protease enterokinase (Lizano et al., 2021). Enterokinase selectively recognizes the trypsinogen substrate sequence DDDDK and cleaves after the lysine residue, thereby exposing the native tethered agonist. Thus, we cloned an Lphn2 construct that included a modified hemagglutinin signal peptide, the P2Y12 N-terminal extracellular sequence (amino acids 1–24), a flexible linker (GGSGGSGGS), the enterokinase recognition site (DYKDDDDK), and the truncated Lphn2-CTF sequence. We tested this construct in a Gβγ-release assay where energy transfer was monitored between the membrane-anchored luminescent donor, GRK3-ct-Rluc8, and the fluorescent acceptor, Gγ-Venus (Hollins et al., 2009). This assay was performed in a HEKΔ7 cell line with targeted deletion of Gα12 and Gα13, as well as Gαs/olf, Gαq/11, and Gαz (Alvarez-Curto et al., 2016) to enable systematic re-introduction of the Gα subunits. As expected, in the absence of Gα subunits no BRET signal was observed; however, when Gα12 or Gα13 was re-introduced to cells expressing the Lphn2 construct there was a significant increase in the BRET signal upon treatment with enterokinase (Figure 2E). This increase was not observed upon co-expression of the receptor with Gαs, Gαi1, or Gαq (Figure 2E and Figure 2—figure supplement 2). This suggests that the increase in cAMP reported previously for the Lphn2 CTF (Sando and Südhof, 2021) may not result from the direct activation of Gαs, but rather from some other form of signaling crosstalk. Alternatively, it is possible that our Lphn2, which was isolated from the P8 hippocampus and lacks exons 19 and 20, may represent a different transcript variant in the brain that activates distinct signaling pathways. In fact, alternative splicing has been shown to affect G protein coupling specificity for several GPCRs, including Lphn3 (Markovic and Challiss, 2009; Röthe et al., 2019). Taken together, these data demonstrate that Lphn2 signals through the G proteins Gα12 and Gα13 in heterologous cells. Having established that these in-cell methods were sufficient to characterize G protein signaling pathways for Lphn2, we next characterized how different mutations in the tethered agonist region affect intracellular signaling. Mutating conserved residues F831A and M835A in the tethered agonist impairs G protein coupling activity Previous studies suggest that the third and seventh residues of aGPCRs are required for tethered agonist-mediated G protein activation (Stoveken et al., 2015). We hypothesized that mutating these residues in Lphn2, phenylalanine (F831), and methionine (M835), to alanine (F831A/M835A) would impair G protein signaling mediated by the tethered agonist (Figure 3A). Like our work with wild-type Lphn2 and Lphn2-CTF, we mutated the tethered agonist residues in both full-length and truncated constructs (Lphn2_F831A/M835A and Lphn2-CTF_F831A/M835A, respectively). Immunoblotting against the C-terminal FLAG-tag confirmed expression in HEK293T cells but showed that Lphn2_F831A/M835A is largely uncleaved (Figure 3B). This is consistent with previous work with Lphn1 showing that mutating the third phenylalanine to an alanine disrupts autoproteolytic cleavage (Araç et al., 2012) and shows that the double mutation (F831A/M835A) in Lphn2 also inhibits cleavage. We also validated that Lphn2_F831A/M835A is expressed on the cell surface at a comparable level as Lphn2 wild-type (Figure 3—figure supplement 1). We then proceeded to test these constructs in our SRE gene expression system (Figure 3A). As hypothesized, both the full-length and truncated Lphn2 had dramatically impaired responses to SRE across varying levels of cDNA transfection. Figure 3 with 2 supplements see all Download asset Open asset Lphn2_F831A/M835A has impaired G protein coupling activity and autoproteolytic cleavage and fails to misdirect proximal CA1 (pCA1) axons to the proximal subiculum (pSub) when misexpressed. (A) Schematic of the mutated tethered agonist for Lphn2_F381A/M835A. The serum response element (SRE) luciferase reporter assay shows that both the full-length Lphn2_F831A/M835A and the Lphn2-CTF_F831A/M835A have impaired signaling (N=3 biological replicates, 9 technical replicates). Means ± SEM; Multiple unpaired t-tests between full-length latrophilin-2 (Lphn2) and Lphn2_F831A/M835A and Lphn2-CTF and Lphn2-CTF_F831A/M835A constructs; ****p<0.0001. (B) Representative immunoblot analysis (N=3) of TA-dead Lphn2 and TA-dead Lphn2-CTF expression in HEK293T cells using a primary antibody against FLAG (1:500, ThermoFisher, PA1-984B). Expected bands for full-length Lphn2_F831A/M835A-FLAG and Lphn2_F831A/M835A-CTF-FLAG are 164 kDa and 72 kDa, respectively. (C) Gβγ-release BRET assay testing SP-P2Y12-EK-Lphn2-CTF_F831A/M835A activation of Gα12 and Gα13 in HEKΔ7 cells (N=3–4 biological replicates, 9–12 technical replicates). SP-P2Y12-EK-Lphn2-CTF signaling is shown for comparison. Means ± SEM; Multiple unpaired t tests between no G protein and G protein conditions; *p<0.05, **p<0.01; ****p<0.0001. (D) Representative images of AAV-DIO-mCh (magenta; mCh expression in a Cre-dependent manner) injections in proximal CA1 (top) and corresponding projections in the subiculum (bottom). (E) Fraction of total axon intensity within proximal, mid, and distal subiculum. Cre: N=5, Cre-Lphn2: N=5 and Cre-Lphn2_F831A/M835A: N=6. Means ± SEM; two-way ANOVA with Sidak’s multiple comparisons test. Injection sites of all subjects are shown in Figure 1—figure supplement 3. Scale bars represent 200 μm. Figure 3—source data 1 Uncropped immunoblot analysis of latrophilin-2 (Lphn2) expression in HEK293T cells. Primary antibody against Flag (1:500, ThermoFisher, PA1-984B), secondary antibody anti-rabbit HRP (1:10,000, ThermoFisher, Cat #31458). Magenta arrows indicate bands of interest for Lphn2-F831A/M835A-CTF-Flag and full-length Lphn2-F831A/M835A-Flag. https://cdn.elifesciences.org/articles/83529/elife-83529-fig3-data1-v2.zip Download elife-83529-fig3-data1-v2.zip Figure 3—source data 2 Lphn2_F831A/M835A has impaired G protein coupling activity and autoproteolytic cleavage and fails to misdirect proximal CA1 (pCA1) axons to the proximal subiculum (pSub) when misexpressed. https://cdn.elifesciences.org/articles/83529/elife-83529-fig3-data2-v2.zip Download elife-83529-fig3-data2-v2.zip To confirm that the reduced SRE response was due to impaired G protein coupling and not simply to impaired proteolysis, we cloned the CTF of our Lphn2_F831A/M835A mutant into our enterokinase-activatable construct. We then tested our construct in the Gβγ-release assay and compared the BRET response to wild-type Lphn2-CTF. Unlike the wild-type receptor, Lphn2-CTF_F831A/M835A did not yield a BRET signal after the re-introduction of any of the G proteins in question (Gαs, Gαi1, Gαq, Gα12, or Gα13) (Figure 3C, Figure 2—figure supplement 2). Taken together, our findings demonstrate that the F831A/M835A mutations in Lphn2 impair tethered agonist-mediated G protein coupling. Tethered agonist activity or autoproteolysis of Lphn2 is required for its cell-autonomous effect in causing proximal CA1 axon mistargeting We next misexpressed Lphn2-F831A/M835A in proximal CA1 to determine if Lphn2 tethered agonist activity or autoproteolysis is required in vivo to direct mistargeting of proximal CA1 axons. Lphn2_F831A/M835A was ectopically expressed at levels similar to that of wild-type Lphn2 and was detected in CA1 axons (Figure 1—figure supplement 1A and B). We injected LV-Cre-P2A-Lphn2_F831A/M835A-FLAG into CA1 of P0 mice, followed by AAV-DIO-mCherry into proximal CA1 of the same mice as adults. The majority of Lphn2_F831A/M835A-expressing proximal CA1 axons targeted the most distal third of the subiculum, like negative control Cre animals (Figure 3D). The fraction of axon intensity in Cre-Lphn2_F831A/M835A animals was significantly lower in the proximal subiculum and significantly higher in the distal subiculum when compared to Cre-P2A-Lphn2 animals (Figure 3E). Proximal CA1 axons in Cre-P2A-Lphn2_F831A/M835A animals showed a similar pattern of targeting to negative control Cre animals (Figure 3—figure supplement 2), although the total fraction of axon intensity was significantly lower in the distal subiculum (Figure 3E). Collectively, these findings suggest that tethered agonist activity or autoproteolysis is required for Lphn2-mediated miswiring of proximal CA1 axons. Mutating residue T829G in the tethered agonist renders Lphn2 cleavage deficient but preserves the ability of the tethered agonist to activate G protein While misexpressing Lphn2_F831A/M835A failed to cause proximal CA1 axons to mistarget to the proximal subiculum, we could not definitively link this result to impaired tethered agonist activity since the Lphn2_F831A/M835A mutant was also resistant to autoproteolytic cleavage (Figure 3B). Since our initial efforts to find a tethered agonist mutant with impaired G protein signaling that retained normal autoproteolytic cleavage were unsuccessful, we designed a construct that rendered Lphn2 resistant to autoproteolytic cleavage but preserved tethered agonist activity. Previous studies showed that replacing threonine-838 in the tethered agonist of Lphn1 or threonine-923 in the tethered agonist of Lphn3 to glycine inhibited autoproteolysis while maintaining prope