Fig 2: Lphn2_T829G impairs autoproteolytic cleavage, retains G protein activity in the truncated receptor, and misdirects axons to the proximal subiculum (pSub) when misexpressed.(A) Representative immunoblot analysis (N=3) of Lphn2_T829G and Lphn2-CTF_T829G expression in HEK293T cells using a primary antibody against FLAG (1:500, ThermoFisher, PA1-984B). Expected bands for full-length Lphn2_T829G-FLAG and Lphn2-CTF_T829G-FLAG are 164 kDa and 72 kDa, respectively. (B) Schematic of the mutated tethered agonist for Lphn2_T829G. The serum response element (SRE) luciferase reporter assay shows that the full-length Lphn2_T829G has impaired SRE levels while the Lphn2_T829G truncated up to the GPS cleavage site has SRE levels comparable to Lphn2_CTF (N=3 biological replicates, nine technical replicates). Means ± SEM; Multiple unpaired t tests between full-length Lphn2 and Lphn2_T829G and Lphn2-CTF and Lphn2-CTF_T829G constructs; *p<0.05; ***p<0.001; ****p<0.0001. (C) Gß?-release BRET assay testing SP-P2Y12-EK-Lphn2-CTF_T829G activation of Ga12 and Ga13 in HEK?7 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<0.001; ****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_T829G: n=5. Means ± SEM; two-way analysis of variance (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 4—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). Green arrows indicate bands of interest for Lphn2-T829G-CTF-Flag and full-length Lphn2-T829G-Flag. Figure 4—source data 2.Lphn2_T829G impairs autoproteolytic cleavage, retains G protein activity in the truncated receptor, and misdirects axons to the proximal subiculum (pSub) when misexpressed.

Fig 3: Exposure of the latrophilin-2 (Lphn2) tethered agonist (TA) promotes intracellular signaling through Ga12/13.(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 Gß?-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 Gß?-Venus to associate with the C-terminus of GPCR kinase 3 (GRK3-ct) (Hollins et al., 2009). (E) Gß?-release BRET assay testing SP-P2Y12-EK-Lphn2-CTF activation of Gas, Gai, Gaq, Ga12, and Ga13 in HEK?7 cells (N=3–4 biological replicates, 9–12 technical replicates). ß2-adrenergic receptor (ß2AR) 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 Gas, Gai and Gaq/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. 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. Figure 2—source data 3.Replicates of the immunoblot assay.Replicate four was used for Figures 2—4.

Fig 4: 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) Gß?-release BRET assay testing SP-P2Y12-EK-Lphn2-CTF_F831A/M835A activation of Ga12 and Ga13 in HEK?7 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. 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.