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.

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 Gα_12/13, and also more weakly to Gα_q, 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 Gα_12/13 as well as Gα_q we tested whether Gα_12/13 or Gα_q was the primary contributor to this response using a selective Gα_q 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 Gα_12/13.

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 Gα_13, as well as Gα_s/olf, Gα_q/11, and Gα_z (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 Gα_s, Gα_i1, or Gα_q (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 Gα_s, 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.

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.

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 (Gα_s, Gα_i1, Gα_q, 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.

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.

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 proper folding of the receptor (Araç et al., 2012; Kordon et al., 2023). Thus, we mutated the analogous threonine-829 in Lphn2 (Lphn2_T829G) and confirmed that Lphn2_T829G was cleavage resistant using immunoblotting (Figure 4A). We also validated that Lphn2_T829G is expressed on the cell surface at a comparable level as Lphn2 wild-type (Figure 3—figure supplement 1).

We next assessed G protein signaling for Lphn2_T829G using our SRE gene expression system with full-length and truncated receptors (Lphn2_T829G and Lphn2-CTF_T829G, respectively) (Figure 4B). Full-length Lphn2_T829G had significantly impaired SRE response compared to wild-type Lphn2, consistent with diminished exposure of the tethered agonist in the absence of cleavage; however, when we tested Lphn2-CTF_T829G, which lacked the entire NTF up to the GPS cleavage site, we observed SRE levels comparable to Lphn2-CTF suggesting that the mutated tethered agonist is fully active if exposed. We, therefore, cloned the CTF of the T829G mutant into our enterokinase-activatable construct and tested BRET signaling following the re-introduction of Gα proteins (Figure 4C). The T829G-CTF retained BRET signaling comparable to wild-type Lphn2-CTF for Gα₁₂ and Gα₁₃, with no discernable BRET response for Gα_s, Gα_i1, or Gα_q (Figure 2—figure supplement 2). Taken together, these data supported that Lphn2_T829G is resistant to autoproteolytic cleavage but maintains a functional tethered agonist.

To assess if autoproteolytic cleavage is required in vivo for Lphn2-mediated proximal CA1 axon mistargeting, we injected Cre-Lphn2_T829G into proximal CA1 of P0 mice, followed by AAV-DIO-mCherry into pCA1 of the same mice as adults. Lphn2_T829G was ectopically expressed at levels similar to wild-type Lphn2 and was detected in CA1 axons (Figure 1—figure supplement 1A and B). Overall, Lphn2_T829G-expressing proximal CA1 axons did not show highly enriched targeting to a specific region of the subiculum, as observed for control proximal CA1 axons or Lphn2 misexpressing proximal CA1 axons, which preferentially target distal and proximal subiculum, respectively (Figure 4E). However, compared to the control there was a significant increase in the fraction of axon intensity in the proximal subiculum in Cre-Lphn2_T829G animals, even though this mistargeting was not as pronounced as seen with wild-type Cre-Lphn2 (Figure 3—figure supplement 2.).

The intermediate gain-of-function phenotypes of misexpressing Lphn2_T829G compared to misexpressing wild-type Lphn2 or tethered agonist-deficient (and non-cleavable) Lphn2 suggest that autoproteolysis is not absolutely required for Lphn2 misexpression-induced miswiring of proximal CA1 axons. The weaker than wild-type overexpression phenotype is likely caused by the decreased G protein signaling of the full-length construct given the more limited exposure of the tethered agonist in the absence of cleavage. The preservation of some signaling activity of Lphn2_T829G is consistent with the ability of its tethered agonist to signal.

We previously showed that misexpression of Lphn2 in distal subiculum target neurons causes proximal CA1 axons to avoid this area, suggesting that Lphn2 acts cell non-autonomously as a repulsive ligand in directing target selection of proximal CA1 axons (Pederick et al., 2021). In the context of repulsive axon guidance, proteolysis has been proposed as a mechanism to disassemble the extracellular binding complex after repulsive signaling, which is necessary for repulsion (Hattori et al., 2000). Are tethered agonist activity and/or autoproteolysis required for Lphn2’s cell non-autonomous role in neural circuit assembly? To test this, we used a strategy to ectopically express Lphn2 in the distal subiculum and trace proximal CA1 axons into the subiculum (Figure 5A; Figure 5—figure supplement 1) identical to the one we previously reported for comparing lentiviruses expressing GFP alone (LV-GFP) or GFP and Lphn2 (LV-GFP-P2A-Lphn2-FLAG; Pederick et al., 2021). At postnatal day 0 (P0), LV-GFP-P2A-Lphn2_F831A/M835A-FLAG and LV-GFP-P2A-Lphn2_T829G-FLAG were injected into distal subiculum, followed by injection of membrane-bound mCherry (AAV-mCherry) into proximal CA1 in the same mice at approximately P42. The P0 lentivirus injection only covers a small fraction of the entire proximal CA1 axon projection, enabling us to assess whether proximal CA1 axons target lentivirus-expressing regions differently from adjacent regions that do not express lentivirus. To observe the relationship between proximal CA1 axon projections and lentivirus-induced regions of the subiculum, we plotted axon signal intensity (mCh) and lentivirus injection site (GFP) from the same animal as height and color, respectively.

We previously reported that GFP alone does not affect the intensity of proximal CA1 axons, whereas GFP-Lphn2 regions have significantly reduced proximal CA1 axon intensity in GFP-Lphn2 positive regions (Pederick et al., 2021; Figure 5B and C; Figure 5—figure supplement 2A and B). When either GFP-Lphn2_F831A/M835A or GFP-Lphn2_T829G were expressed in the distal subiculum, we also observed a significant decrease in axon intensity in GFP positive regions compared to GFP (Figure 5D, E and F and Figure 5—figure supplement 2C and D). This decrease was not significantly different from GFP-Lphn2 animals (Figure 5F). These findings suggest that neither tethered agonist activity nor autoproteolysis is required for Lphn2’s cell-non-autonomous role as a ligand in the neural circuit assembly.

Upon aGPCR biosynthesis, the conserved GAIN domain undergoes autoproteolytic cleavage at the GPS to generate N- and C-terminal fragments that remain non-covalently bound during trafficking to the cell surface (Araç et al., 2012). Crystal structures of GAIN domains from Lphn1 and ADGRB3 (BAI3) (Araç et al., 2012), ADGRG1 (GPR56) (Salzman et al., 2016), and ADGRG6 (GPR126) (Leon et al., 2020), revealed that in an intact aGPCR, the tethered agonist is buried as a β-strand in the GAIN domain, forming an extensive network of conserved hydrogen bonds and hydrophobic side chains. This suggests that when the complex between aGPCR’s NTF and CTF remains intact the tethered agonist is inaccessible for engagement with its binding site in the 7TM domain. However, several studies have reported that naturally cleavage-resistant aGPCRs can still function (Liebscher et al., 2014; Wilde et al., 2016). This suggests that the tethered agonist can in fact interact with the 7TM independent of cleavage, although the efficacy of this interaction is likely to be diminished, as we see in the present study. Thus, the relative contributions and/or necessity of autoproteolytic cleavage and the tethered agonist to aGPCR activity remain an area of active study.

Recent efforts reported the structures of eight aGPCRs, seven of which were truncated up to the GPS (Barros-Álvarez et al., 2022; Ping et al., 2022; Qu et al., 2022; Xiao et al., 2022). While most discussions from the structural aGPCR studies argued that NTF dissociation is required for tethered agonist interaction with the receptor, the structure of autoproteolysis-deficient ADGRF1 supported the possibility of the cleavage-independent manner of receptor activation (Qu et al., 2022). The density for the tethered agonist of ADGRF1 was well-resolved and bound in an α-helical structure within the orthosteric site of the 7TM bundle. This interaction was like that of the cleaved structures, and ADGRF1 was also observed to be bound to a miniG_i1, supporting that receptor cleavage and tethered agonist exposure are not absolutely required for G protein coupling. One caveat, however, is that there is little density for the NTF in this structure, suggesting that the structure obtained may result from a fraction of receptor where cleavage has still occurred and the NTF has dissociated.

As mentioned above, not all aGPCRs are auto-proteolytically cleaved; therefore, activation cannot be fully dependent on tethered agonist exposure through the removal of the NTF (Kishore et al., 2016; Liebscher et al., 2022). In this regard, it is possible that full-length aGPCRs exist in multiple conformational states that include receptor molecules in which the tethered agonist is unmasked from the GAIN domain. In fact, molecular dynamics (MD) simulations of spontaneous tethered agonist exposure were recently reported for five intact aGPCR homologs (ADGRB3, ADGRE2, ADGRE5, ADGRG1, and Lphn1) (Beliu et al., 2021). Here, the authors show that tethered agonist exposure occurs due to the high intrinsic flexibility of the GAIN domain. They also used biorthogonal labeling of conserved positions within the tethered agonist to show that large portions (+6 residues) of the tethered agonist can become solvent accessible in the context of the GAIN domain. They argue that tethered agonist exposure likely occurs in a stepwise mechanism where the tethered agonist is uncovered along its N→C axis. Thus, it is possible that an intact complex of aGPCR’s NTF and CTF could unmask the tethered agonist sufficiently for interaction with the 7TM, resulting in receptor activation.

The ability of tethered agonist exposure to occur in intact aGPCRs could provide an explanation for why our Lphn2_T829G mutant displays a partial axon mistargeting phenotype (Figure 4). Even though the Lphn2_T829G mutant cannot undergo autoproteolytic cleavage, it still retains a functional tethered agonist that is able to initiate G protein signaling if transiently unmasked. This is likely why activation of full-length Lphn2_T829G is less robust than the wild-type Lphn2, which can more readily unmask the TA. We also cannot rule out the possibility that a small amount of cleavage, although undetectable in the HEK cells immunoblotting, nonetheless contributes to the partial mistargeting phenotype in vivo.

Like the Lphn2_T829G mutant, Lphn2_F831A/M835A cannot undergo autoproteolytic cleavage. However, this mutant also has impaired G protein coupling activity even with full exposure of the TA. This explains why we observed close-to-normal axon targeting when we overexpressed Lphn2_F831A/M835A in proximal CA1 (Figure 3). Even if the tethered agonist of Lphn2_F831A/M835A becomes unmasked it still cannot initiate tethered agonist-mediated receptor. However, given the statistically significant difference in total axon intensity in dSub between control and Lphn2_F831A/M835A (Figure 3E), we cannot rule out some residual tethered agonist-independent, G protein-mediated signaling or an Lphn2 function independent of G protein signaling, at least in the context of overexpression.

How could Lphn2-mediated G protein signaling in the CA1 axons lead to axon repulsion? We show here that Lphn2 primarily signals through Gα₁₂ and Gα₁₃ in heterologous cells (Figure 2E). Gα₁₂/Gα₁₃ are known to regulate Rho GTPase; for example, Gα₁₃ binds to and activates p115RhoGEF, an exchange factor for and activator of the small GTPase RhoA (Kozasa et al., 2011). RhoA activation is known to cause growth cone collapse and neuronal process retraction via its regulation of the actin-myosin contractility (Luo, 2002; Spillane and Gallo, 2014). Thus, if Lphn2 coupling to Gα12/Gα13 also applies to CA1 neurons, as suggested by our results in heterologous cells, Gα12/Gα13→RhoGEF→RhoA may be a plausible pathway for Lphn2 to mediate its function as a receptor for axon repulsion.

Interestingly, neither autoproteolytic cleavage nor tethered agonist activity is required for Lphn2 to act cell non-autonomously as a repulsive ligand in subiculum target neurons (Figure 5). This suggests that cleavage of Lphn2 is not required for repulsion in this context and implies that another mechanism mediates the disassembly of the extracellular binding complex, which is required for retracting axons to pull away from the targets. Other potential mechanisms to disassemble the extracellular binding complex include Ten3 cleavage (teneurins are known to also undergo proteolytic cleavage at its extracellular domain; Sita et al., 2019), endocytosis of the adhesion complex as in the case of ephrin/Eph receptor (Egea and Klein, 2007), or forces produced by actin-myosin contractility in axon terminals induced by repulsive signaling. Indeed, since Lphn2 also acts as a repulsive ligand and Ten3 as a repulsive receptor, extracellular binding of Lphn2 to Ten3 should also trigger a repulsive response in Ten3+ axon terminals, but the signaling mechanism is completely unknown. Future studies on the mechanisms that disassemble the extracellular complex and intracellular signaling in the axon downstream of Ten3 will increase our understanding of how the interaction of these two molecules can lead to repulsive outcomes.

Dulbecco’s Modified Eagle Medium (DMEM), high glucose, and penicillin-streptomycin (P/S) (10,000 U/mL) were purchased from Gibco (ThermoFisher Scientific, Waltham, MA). Fetal bovine serum (FBS), 0.5% trypsin, and Dulbecco’s phosphate-buffered saline (DPBS) were purchased from Corning (Fisher Scientific, Waltham, MA). Opti-MEM reduced serum medium, no phenol red, and Lipofectamine 2000 transfection reagent was purchased from Invitrogen (ThermoFisher Scientific). FuGENE transfection reagent was purchased from Promega (Madison, WI). RIPA buffer was purchased from Sigma-Aldrich (St. Louis, MO). Triton lysis buffer consisted of 0.11 M Tris-HCl powder, 0.04 M Tris-base powder, 75 mM NaCl, 3 mM MgCl₂, and 0.25% Triton X-100 pure liquid. The 3 X Firefly Assay Buffer was freshly prepared in Triton lysis buffer and contained 15 mM DTT, 0.6 mM coenzyme A (MedChemExpress, Monmouth Junction, NJ), 0.45 mM ATP (MedChemExpress, Monmouth Junction, NJ), and 0.42 mg/mL firefly D-luciferin (NanoLight Technology). Renilla Salts buffer consisted of 45 mM Na₂EDTA, 30 mM Na Pyrophosphate, and 1.425 M NaCl. The 3 X Renilla Assay Buffer was freshly prepared in Renilla Salts and contained 0.06 mM PTC124 in DMSO (MedChemExpress) and 0.01 mM coelenterazine-h (NanoLight Technologies, Pinetop, AZ). For the BRET assays, enterokinase, light chain, was obtained from New England Biolabs (Ipswich, MA), isoproterenol and quinpirole from Sigma Aldrich, and endothelin 1 (ET-1) from Tocris Bioscience (Bristol, United Kingdom). YM-254890 was purchased from AdipoGen Life Sciences (San Diego, CA). Impermeant Janelia Fluor 646 conjugated to benzyl guanine was a kind gift from Dr. Luke Lavis (Howard Hughes Medical Institute Janelia Research Campus).

Lphn2 was amplified from cDNA isolated from the P8 mouse hippocampus. Sanger sequencing confirmed that exons 19 and 20 were excluded from the amplified Lphn2 (Refer to NCBI Reference Sequence: NM_001081298.2 for exon annotation). This cDNA was used as a polymerase chain reaction (PCR) template to make the Lphn2 constructs used in this study. All cDNA constructs were assembled in a pCDNA3.1+ vector by Gibson assembly using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs). Sequences were confirmed with the Genewiz sequencing service (South Plainfield, NJ). Plasmid DNA constructs are available upon request.

HEK293T cells (American Type Culture Collection, Manassas, VA; RRID:CVCL_0063) and HEK293 cells with targeted deletion via CRISPR-Cas9 of GNAS, GNAL, GNAQ, GNA11, GNA12, GNA13, and GNAZ (HEKΔ7) (Alvarez-Curto et al., 2016) were maintained in high-glucose DMEM supplemented with 10% FBS and 1% P/S at 37 °C in a 5% CO₂ humidified incubator. Cell authentication was not performed as the cells were obtained directly from the supplier. PCR-based mycoplasma testing was performed routinely using ATCC mycoplasma testing services. Cell viability was assessed for each passage using the Countess II automated cell counter (ThermoFisher Scientific).

HEK293T cells were detached for 2–3 min using 0.5% trypsin and then plated at a density of 350,000 cells/mL in a six-well culture plate. After 24 hr, the cells were transfected using FuGENE transfection reagent (8 μL/2 μg cDNA) and Opti-Mem with receptor cDNA (2 μg). After 24 hr, cells were placed on ice and incubated in 500 μL RIPA buffer for 30 min. Following this incubation, cells were scraped from the culture plate and moved into 1.5 mL microcentrifuge tubes. Cells were then spun at 15,000 × g in a 4 °C benchtop centrifuge to pellet debris. After centrifugation, 50 μL of the supernatant was transferred into a fresh microcentrifuge tube and combined with 50 μL 2 X SDS Laemmli sample buffer (Sigma-Aldrich). In preparation for immunoblot analysis, a 20 μL sample was run on an SDS-PAGE gel (Mini-PROTEAN TGX, 4–15%, Bio-Rad Laboratories, Inc, Hercules, CA) prior to transfer to a PDVF membrane (Immobilon-P Membrane, Merck Millipore Ltd., Burlington, MA). The membrane was then incubated in a 5% milk tris-buffered saline with 0.1% tween-20 (TBS-T) solution for 1 hr at RT with gentle rotation. The membrane was washed five times 5 min in TBS-T prior to overnight incubation at 4 °C with 1° anti-FLAG antibody (1:500, ThermoFisher, PA1-984B; RRID:AB_347227). The next morning, the membrane was washed five times 5 min in TBS-T. The membrane was then incubated for 1 hr at RT with 2° anti-rabbit HRP antibody (1:10,000, ThermoFisher, Cat #31458;RRID:AB_228213). The membrane was washed five times 5 min in TBS-T prior to visualization with SuperSignal West Pico Chemiluminescent Substrate (Fisher Scientific) using the Azure c600 Gel Imaging System (Azure Biosystems, Dublin, CA).

In preparation for transfection, HEK293T cells were detached for 2–3 min using 0.5% trypsin and then seeded at a density of 400,000 cells/mL in a 12-well culture plate. After 24 hr, the cells were co-transfected using Lipofectamine 2000 (2.5 μL/1 μg cDNA) and Opti-Mem with receptor cDNA (10–600 ng), gene reporter cDNA (600 ng), and empty vector pCDNA5/FRT to balance the total amount of cDNA up to 1200 ng. After 6 hr with the transfection reagent, the media was volume exchanged to serum-free DMEM supplemented with 1% P/S (~18 hr serum starvation).

After 24 hr, the media was aspirated from the cells and each well was gently rinsed with DPBS. Cells were then mechanically detached using 275 μL DPBS and 80 μL of the resuspension was distributed in triplicate to a 96-well black/white isoplate (Perkin Elmer Life Sciences). Next, 40 μL of 3 X Firefly Assay Buffer was added to each well. The emission was then read at 535 nm after 10 min incubation using a PHERAstar FS microplate reader (BMG LABTECH, Ortenberg, Germany). Next, 60 μL 3 X Renilla Assay Buffer was added to each well. The emission was then read at 475 nm after 10 min incubation using a PHERAstar FS microplate reader. For assays using the Gαq-inhibitor YM-254890, the cell media was exchanged to DMEM containing 1 μM YM-254890 approximately 6 hr after transfection.

In preparation for transfection, HEKΔ7 cells were detached for 2–3 min using 0.5% trypsin and then seeded at a density of 400,000 cells/mL in a 12-well culture plate. After 24 hr, the cells were co-transfected using Lipofectamine 2000 (2.5 μL/1 μg cDNA) and Opti-Mem with receptor cDNA (200 ng), Gα (720 ng), Gβ1 (250 ng), Gγ2-Venus (250 ng), membrane-anchored GRK3ct-Rluc8 (50 ng), and empty vector pCDNA5/FRT to balance the total amount of cDNA up to 1470 ng. After 24 hr transfection, cells were washed with DPBS before being re-suspended in 400 μL BRET buffer (DPBS containing 5 mM glucose). Next, 45 μL of the resuspension was distributed to six wells of a 96-well OptiPlate black-white plate (Perkin Elmer Life Sciences, Waltham, MA). Cells were then incubated for 10 mins with 10 μL coelenterazine-h (final concentration 5 μM) before ligand addition to reach a final well volume of 100 μL. Donor (Rluc8) and acceptor (mVenus) emission was read using a PHERAstar FS microplate reader at 485 nm and 525 nm, respectively. The BRET ratio was then measured by dividing the 525 emissions by the 485 emissions. The drug-induced BRET ratio was then calculated by subtracting the buffer BRET for each condition.

In preparation for transfection, HEK293T cells were detached for 2–3 min using 0.5% trypsin and then seeded at a density of 900,000 cells/well in a six-well culture plate. After 24 hr, the cells were transfected using FuGENE transfection reagent (8 μL/2 μg cDNA) and Opti-Mem with SNAPfast-tagged receptor cDNA (2 μg). After 24 hr, cells were incubated for 30 min with 500 µL 1 µM impermeant Janelia Fluor 646 conjugated to benzyl guanine was dissolved in DMEM containing 10% FBS and 1% P/S. Cells were then washed three times with complete DMEM and once with DPBS prior to resuspension in 500 µL DPBS. Next, 100 μL of resuspension was added to three wells of a 96-well OptiPlate black plate (Perkin Elmer Life Sciences, Waltham, MA). The emission was then read using the filter 640/680 at a gain of 1000 using a PHERAstar FS microplate reader (BMG LABTECH, Ortenberg, Germany).

All procedures followed animal care and biosafety guidelines approved by Stanford University’s Administrative Panel on Laboratory Animal Care (APLAC 14007) and Administrative Panel on Biosafety (APB-3669-LL120) in accordance with NIH guidelines. Both male and female mice were used, and mice were group housed on a 12 hr light/dark cycle with access to food and water ad libitum. CD-1 mice from Charles River Laboratories were used for all experiments. The total number of mice injected and screened for each experiment is as follows: Figure 1: LV-Cre, 101, LV-Cre-P2A-Lphn2-FLAG, 60; Figure 3: LV-Cre-P2A-Lphn2_F831A/M835A-FLAG, 86; Figure 4: LV-Cre-P2A-Lphn2_T829G-FLAG, 102; and Figure 5: LV-GFP-P2A-Lphn2_F831A/M835A-FLAG, 79 and LV-GFP-P2A-Lphn2_T829G-FLAG, 56.

All lentivirus constructs expressing Cre, GFP, Lphn2, Lphn2_F831A/M835A, or Lphn2_T829G were made by inserting corresponding cDNA into the LV-UbC plasmid (Pederick et al., 2021) with a P2A sequence between the two ORFs. Cre and GFP were amplified from LV-UbC-GFP-Cre and full-length Lphn2 cDNA was isolated from a cDNA library made from mRNAs from the P8 mouse hippocampus. GFP and Lphn2 were inserted into LV-UbC with a Gibson assembly cloning kit (NEB E5510S). The Lphn2_F831A/M835A and Lphn2_T829G mutations were made using Q5 mutagenesis (NEB, E0552S). All plasmids were sequenced and verified before the virus was produced. All custom lentiviruses were generated by transfecting 36 10 cm plates (HEK293T) with four plasmids (4.1 R, RTR2, VSVg, and transfer vector containing gene of interest). Medium was collected 48 hr later and centrifuged at 8400 relative centrifugal force (rcf) for 18 hr at 4 °C. Viral pellets were dissolved with PBS and further purified with a 20% sucrose gradient centrifugation at 80,000 rcf for 2 hr.

P0 mice were anesthetized using hypothermia. CA1 injections were 1.0 mm lateral, 0.85 mm anterior, and 0.8 mm ventral from lambda and subiculum injections were 1.3 mm lateral, 0.45 mm anterior, and 0.8 mm ventral from lambda. 100 nl of lentivirus was injected at 100  nl/min at the following titers: LV-Cre (7 × 10¹² copies per ml), LV-Cre-P2A-Lphn2-FLAG (2.4 × 10¹² copies per ml), LV-Cre-P2A-Lphn2_F831A/M831A-FLAG (2.24 × 10¹² copies per ml) and LV-Cre-P2A-Lphn2_T829G-FLAG (1.2 × 10¹³ copies per ml), LV-GFP (6 × 10¹² copies per ml), LV-GFP-P2A-Lphn2-FLAG (5 × 10¹² copies per ml), LV-GFP-P2A-Lphn2_F831A/M831A-FLAG (3.6 × 10¹² copies per ml), LV-GFP-P2A-Lphn2_T829G-FLAG (9 × 10¹² copies per ml).

Injections of AAV8-EF1a-DIO-ChR2-mCh (2 × 10¹² copies per ml, Neuroscience Gene Vector and Virus core, Stanford University) and AAV8-CaMKIIa-ChR2-mCh (2 × 10¹² copies per ml, Neuroscience Gene Vector and Virus core, Stanford University) were performed at about P42. Mice were anesthetized using isoflurane and mounted in stereotactic apparatus (Kopf). Coordinates for proximal CA1 were 1.4 mm lateral and 1.25 mm posterior from bregma, and 1.12 mm ventral from the brain surface. Virus was iontophoretically injected with current parameters 5 µA, 7 s on, 7 s off, for 2 min, using pipette tips with an outside perimeter of 10–15 μm. Mice were perfused about 2 weeks later and processed for immunostaining as described below.

Mice were injected with 2.5% Avertin and were transcardially perfused with PBS followed by 4% paraformaldehyde (PFA). Brains were dissected and post-fixed in 4% PFA overnight, and cryoprotected for about 24 hr in 30% sucrose. Brains were embedded in Optimum Cutting Temperature (OCT, Tissue-Tek), frozen in dry ice-cooled isopentane bath, and stored at –80 °C until sectioned. 60 μm thick floating sections were collected in PBS +0.02% sodium azide and stored at 4 °C. Sections were incubated in the following solutions at room temperature unless indicated: 1 hr in 0.3% PBS/Triton X-100 and 10% normal donkey serum, two nights in the primary antibody at 4 °C in 0.3% PBS/Triton X-100 and 10% normal donkey serum, 3 × 15 min in 0.3% PBS/Triton X-100, overnight in secondary antibody +DAPI (1:10,000 of 5 mg/ml, Sigma-Aldrich) in 0.3% PBS/Triton X-100 and 10% normal donkey serum, 2 × 15 min in 0.3% PBS/Triton X-100, and 15 min in PBS. Sections were mounted with Fluoromount-G (SouthernBiotech). Primary antibodies used were rat anti-mCherry (1:1000, ThermoFisher, M11217, RRID:AB_2536611), rabbit anti-Cre (1:500, Synaptic Systems, 257 003, RRID:AB_2619968), chicken anti-GFP (1:2500, Aves Labs, GFP-1020, RRID:AB_10000240) goat anti-FLAG (1:3000, Abcam, ab95045, RRID:AB_10676074) and rabbit anti-Lphn2 (1:500, Novus Biologicals, NBP2-58704). Secondary antibodies conjugated to Alexa 488, Alexa 568, or Cy3 (Jackson ImmunoResearch) were used at 1:500 from 50% glycerol stocks.

Mice were only included if they passed the following criteria: (1) AAV injection site must be in proximal CA1 (most proximal 30%), (2) lentivirus injections sites must be in CA1 and not in the subiculum (Figures 1, 3 and 4) or lentivirus injections must be in the distal subiculum (Figure 5), (3) proximal CA1 axons must overlap with lentivirus injection site in the subiculum (Figure 5). All mice that fulfilled these criteria are reported in Figures 1 and 3—5 and were included in quantifications. Images of injection sites (5x magnification) and projections (10x magnification) were acquired for every other 60 μm sagittal section using a Zeiss epifluorescence scope. Due to variations in injection sites within each mouse, exposure was adjusted for each mouse to avoid saturation. Fluorescence intensity measurements on unprocessed images were taken using FIJI and data processing was performed using MATLAB.

For injection site quantification, a 30-pixel-wide segmented line was drawn from proximal CA1 to distal CA1 using the DAPI signal as a guide. For projection quantification in the subiculum, a 200-pixel-wide segmented line was drawn from the proximal subiculum to the distal subiculum through the cell body layer using only DAPI as a guide. From this point, injection site and projection images were processed the same. Segmented lines were straightened using the ‘Straighten’ function, background subtraction was performed using the ‘Subtract’ function and intensity values were measured using the ‘Plot Profile’ command (FIJI). For injections that labeled both CA2 and proximal CA1, CA2 axons were present near the distal border of CA1 and spilled into the proximal subiculum. These axons had their intensity set to zero by using area selection and the clear function (FIJI). The intensity plots were resampled into 100 equal bins using a custom MATLAB code.

For trace quantification in Figures 1, 3 and 4 the axon intensity was combined for all sections by summing all intensity values at each binned position. To calculate the fraction of axon intensity across proximal, mid, and distal subiculum the total axon intensity in bins 1–33, 34–66, and 67–100 were summed, respectively. The summed value from each of these regions was then divided by the total sum of axons from bins 1–100 to obtain the fraction of axons intensity within the proximal, mid, and distal subiculum. Fractions of axon intensities were compared using a two-way ANOVA with Sidak’s multiple comparisons tests using Prism 9 (GraphPad). The mean position of the injection sites was calculated by generating a summed intensity trace as above and then multiplying the intensity value by the bin position, summing across the entire axis, and dividing by the sum of the intensity values. Representative images (Figures 1, 3 and 4) were taken using a Zeiss LSM 780 confocal microscope (20×magnification, tile scan, max projection).

In Figure 5, the experimental and data analysis procedures were identical to Pederick et al., 2021 and, therefore, we used LV-GFP and LV-GFP-P2A-Lphn2-FLAG from that study to compare with LV-GFP-P2A-Lphn2_F831A/M831A-FLAG and LV-GFP-P2A-Lphn2_T828G-FLAG data generated from this study.

To quantify average axon intensity in GFP+ and adjacent GFP– regions in subiculum targets (Figure 5F), we restricted the analysis to the most distal 20% of the subiculum. To determine the GFP+ region we identified the intensity-weighted central row using the summed fluorescence of each row and determined the minimal symmetric window of rows around the central row that encompassed at least 50% of the total intensity in the restricted GFP image. This defined a rectangle in the original image that we designated as the GFP+ region. We then computed the mean fluorescence intensity in this region for the mCh channel. We used the two rows above and below (lateral and medial) the designated GFP+ region as the adjacent GFP– region and computed the mean mCh fluorescence across these four rows. To determine mCh fluorescence differences in GFP+ versus GFP– regions, we divided the mCh intensity in the GFP+ region by the mCh intensity in the GFP– region for each mouse (i.e. GFP+/GFP–). mCh fluorescence intensity GFP+/GFP– was compared across groups using a one-way ANOVA with Tukey’s multiple comparisons tests using Prism 9 (GraphPad). Three-dimensional mountain plots were generated using the ‘surf’ function.

P0 neonatal injections into CA1 and immunostaining were performed as stated above. 16-bit images were acquired with a Zeiss Axio Imager Z2 confocal microscope. All conditions were imaged with equal settings. For mean intensity quantification of FLAG and GFP a 50- and 20-pixel wide segmented line was drawn through the molecular layer and cell body layer of CA1, respectively, using the DAPI signal as a guide. The mean intensity of FLAG and GFP was calculated using the ‘Measure’ function.

From this point, only the section with the highest mean GFP expression was used from each animal. The FLAG mean intensity value was divided by the GFP mean intensity value to determine the FLAG/GFP mean intensity. FLAG/GFP mean intensities across conditions were compared using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test in Prism 9 (GraphPad).

Technical replicates are defined here as repeated measurements of the same sample while biological replicates are defined as measurements of biologically distinct samples.