Latrophilins: Overview

Latrophilins (Lphns, also named CIRLs or CLs) are evolutionarily conserved adhesion-GPCRs (aGPCRs) that are encoded by three genes (ADGRL1-3 in humans, Adgrl1-3 in mice)^1,2. Lphns contain N-terminal extracellular adhesion domains (a lectin-like, an olfactomedin-like, and a ‘hormone-binding’ domain) and a canonical GAIN domain characteristic of aGPCRs that undergoes autoproteolysis³. These domains are followed by a typical 7-transmembrane region GPCR module and a large cytoplasmic sequence (see Figure). Lphn transcripts are subject to extensive alternative splicing that affects both their extracellular and intracellular sequences and produces alternative cytoplasmic tails for all latrophilins (see Figure)⁴. The C-terminal alternative splicing of Lphn3, and possibly other Lphns, regulates its interaction with G-proteins and with postsynaptic scaffolds and is activity-dependent⁴.

Lphns are expressed in adult mammals most highly in brain where they localized, at least in part, to synapses^5-7. Lphn1, and possibly Lphn2 and Lphn3 as well, are present in both excitatory and inhibitory synapses. In these synapses, Lphn1 and its ligand teneurin-3 form nanoclusters occupying ~20% of a synaptic junction, which is similar to observations made for other synaptic adhesion molecules^7,8.

Lphns bind to multiple extra- and intracellular interacting proteins. Extracellularly, their interaction with FLRTs and teneurins has been validated by crystal and cryo-EM structures (see Figure)^9-13. FLRTs bind to the olfactomedin-like domain of Lphns, while teneurins bind to their lectin-like domain. FLRTs and teneurins represent families of adhesion molecules that, as discussed below, likely also bind to other adhesion molecules besides Lphns. Cryo-EM structures revealed that Lphns can simultaneously bind to FLRTs and to teneurins that form constitutive homodimers^9-13. As a result, the teneurin-Lphn-FLRT complex can potentially form an extended lattice in the synaptic cleft.

Intracellularly, the splice variants of all Lphns containing the conserved C-terminal ‘LVSTL*’ sequence (see domain structure figure above) interact with the PDZ-domain of Shank proteins, which are postsynaptic scaffold proteins^14,15. Both this and the other C-terminal splice variants of Lphns comprise long sequences that likely bind to other cytoplasmic proteins, but these proteins have not yet been characterized.

Functionally, Lphns have been shown to control synapse numbers in the CA1 region of the hippocampus but their roles in other circuits, brain regions and tissues are poorly investigated. In CA1 region pyramidal neurons, Lphn2 is selectively essential for excitatory synapses formed in the S. lacunosum-moleculare by inputs from the entorhinal cortex, whereas Lphn3 is selectively essential for excitatory synapses formed in the S. oriens and S. radiatum by Schaffer-collateral inputs from the CA3 region^5,6,16. Lphn1, in turn, appears to be only required for somatic inhibitory synapses⁷. The functions of Lphn2 and Lphn3 require its GPCR activity¹⁶, and at least for Lphn3 both the teneurin- and the FLRT-binding sites are functionally necessary⁶. Although it was postulated based on cryo-EM structures that Lphns are activated by its tethered agonist that is exposed by GAIN-domain cleavage^17,18, GAIN-domain cleavage of Lphn3 is not required for synaptic function⁶. As GPCRs, Lphns are coupled to multiple Ga-proteins, with the C-terminal Lphn3 splice variant that is selectively required for its synaptic function preferentially coupled to GaS, resulting in cAMP synthesis⁴. Indeed, selectively suppressing cAMP levels in postsynaptic specializations causes a loss of synapses, consistent with a Lphn-induced cAMP-signaling pathway in synapse formation¹⁹.

Knowledge gaps. Lphns are multifunctional GPCRs that nucleate large multimolecular assemblies at synapses and likely elsewhere. Multiple key questions remain unanswered:

a)    What is the precise composition of Lphn-based synaptic complexes that drive synapse formation, and do they differ between Lphn isoforms and synapse types?

b)    What are the relative ligand-binding affinities of different Lphns, FLRTs, and teneurins, and could their differential affinities determine synapse specificity?

c)     What is the mechanism of Lphn-induced signaling that mediates the synapse assembly driven by their GPCR activity – in particular, what signaling scaffolds do Lphns recruit to their C-termini?

d)    Do Lphns generally function in both excitatory and inhibitory synapses, and if so, do they act by similar mechanisms despite the fact that the postsynaptic scaffolds of excitatory and inhibitory synapses are different with no apparent overlap?

e)    Why are teneurin- and FLRT-binding both required for Lphn3 function at synapses, and does this also apply to other Lphns?

f)      Are FLRTs and their Unc5 interactors also involved in synapse formation similar to Lphns and teneurins?

g)    What is the function of different Lphn splice variants, for example variants that do not interact with Shank proteins or that poorly bind to teneurins¹⁹?

h)    What are the non-neuronal functions of Lphns?

These and many other questions need to be addressed for a better understanding of these crucial aGPCRs.

References

1.     Sugita, S., Ichtchenko, K., Khvotchev, M., and Südhof, T.C. (1998) α-latrotoxin receptor CIRL/latrophilin 1 (CL1) defines an unusual family of ubiquitous G-protein linked receptors. J. Biol. Chem. 273, 32715-32724.

2.     Ichtchenko K, Bittner MA, Krasnoperov V, Little AR, Chepurny O, Holz RW, Petrenko AG (1999) A novel ubiquitously expressed alpha-latrotoxin receptor is a member of the CIRL family of G-protein-coupled receptors. J. Biol. Chem. 274, 5491-5498.

3.     Arac, D., Boucard, A.A., Bolliger, M.F., Nguyen, J., Soltis, M., Südhof, T.C., and Brunger, A.T. (2011) A Novel Evolutionarily Conserved Domain of Cell-Adhesion GPCRs Mediates Autoproteolysis. EMBO J. 31, 1364-1378.

4.     Wang, S., DeLeon, C., Sun, W., Quake, S.R., Roth, B.L., and Südhof, T.C. (2023) Alternative Splicing of Latrophilin-3 Controls Synapse Formation. Nature, in press.

5.     Anderson, G., Maxeiner, S., Sando, R., Tsetsenis, T., Malenka, R.C., and Südhof, T.C. (2017) Postsynaptic Latrophilin-2 Mediates Target Recognition in Entorhinal-Hippocampal Synapse Assembly. J. Cell Biol. 216, 3831-3846.

6.     Sando, R., Jiang, X., and Südhof, T.C. (2019) Latrophilin GPCRs direct synapse specificity by coincident binding of FLRTs and teneurins. Science 363, pii: eaav7969.

7.     Matúš, D., Lopez, J.M., Sando, R.C., and Südhof, T.C.  The Essential Role of Latrophilin-1 Adhesion GPCR Nanoclusters in Inhibitory Synapses (2023) BioRX Preprint  DOI: https://doi.org/10.1101/2023.10.08.561368

8.     Zhang, X., Lin, P.Y., Liakath-Ali, K, and Südhof, T.C. (2022) Teneurins Assemble into Presynaptic Nanoclusters that Promote Synapse Formation via Postsynaptic Non-Teneurin Ligands. Nature Comm. 13, 2297.

9.     Lu, Y.C., Nazarko, O., Sando, R., Salzman, G., Südhof, T.C., and Araç, D. (2015) Crystal Structure of the FLRT3-LPHN3 complex reveal specific protein-protein interactions.  Structure 23, 1678-1691.

10.  Li, J., Shalev-Benami, M., Sando, R., Jiang, X., Kibrom, A., Wang, J., Leon, K., Katanski, C., Nazarko, O., Lu, Y.C., Südhof, T.C., Skiniotis, G., and Araç, D. (2018) Structural basis for teneurin function in circuit-wiring: A toxin motif at the synapse. Cell 173, 735-748.

11.  Jackson VA, Meijer DH, Carrasquero M, van Bezouwen LS, Lowe ED, Kleanthous C, Janssen BJC, Seiradake E (2018) Structures of Teneurin adhesion receptors reveal an ancient fold for cell-cell interaction. Nat. Commun. 9, 1079. 

12.  Li, J., Xie, Y., Cornelius, S., Jiang, X., Sando, R., Kordon, S., Pan, M., Leon, K., Südhof, T.C., Zhao, M., and Araç, D. (2020) Alternative splicing controls teneurin-latrophilin interaction and synapse specificity by a shape-shifting mechanism. Nature Comm. 11, 2140.

13.  Del Toro D, Carrasquero-Ordaz MA, Chu A, Ruff T, Shahin M, Jackson VA, Chavent M, Berbeira-Santana M, Seyit-Bremer G, Brignani S, Kaufmann R, Lowe E, Klein R, Seiradake E (2020) Structural Basis of Teneurin-Latrophilin Interaction in Repulsive Guidance of Migrating Neurons. Cell 180, 323-339.

14.  Tobaben, S., Südhof, T.C., and Stahl B. (2000) The G protein-coupled receptor CL1 interacts directly with proteins of the Shank family. J. Biol. Chem. 275, 36204-36210.

15.  Kreienkamp HJ, Zitzer H, Gundelfinger ED, Richter D, Bockers TM (2000) The calcium-independent receptor for alpha-latrotoxin from human and rodent brains interacts with members of the ProSAP/SSTRIP/Shank family of multidomain proteins. J. Biol. Chem. 275, 32387-32390

16.  Sando, R., and Südhof, T.C. (2021)  Latrophilin GPCR Signaling Mediates Synapse Formation. E-Life 10, e65717.

17.  Barros-Álvarez X, Nwokonko RM, Vizurraga A, Matzov D, He F, Papasergi-Scott MM, Robertson MJ, Panova O, Yardeni EH, Seven AB, Kwarcinski FE, Su H, Peroto MC, Meyerowitz JG, Shalev-Benami M, Tall GG, Skiniotis G (2022) The tethered peptide activation mechanism of adhesion GPCRs. Nature 604, 757-762. 

18.  Qian Y, Ma Z, Liu C, Li X, Zhu X, Wang N, Xu Z, Xia R, Liang J, Duan Y, Yin H, Xiong Y, Zhang A, Guo C, Chen Z, Huang Z, He Y (2022) Structural insights into adhesion GPCR ADGRL3 activation and G_q, G_s, G_i, and G₁₂ coupling. Mol. Cell 82, 4340-4352.

19.  Sando, R., Ho, M.L., Liu, X., and Südhof, T.C. (2022) Engineered Synaptic Tools Reveal Localized cAMP Signaling in Synapse Assembly. J. Cell Biol. 221, e202109111.

Wang Y, Cao Y, Hays CL, Laboute T, Ray TA, Guerrero-Given D, Ahuja AS, Patil D, Rivero O, Kamasawa N, Kay JN, Thoreson WB, Martemyanov KA (2021) Adhesion GPCR Latrophilin 3 regulates synaptic function of cone photoreceptors in a trans-synaptic manner. Proc. Natl. Acad. Sci. USA 118, e2106694118.

Lphn-Teneurin Complexes

Properties. Teneurins are large type II membrane proteins comprised of a relatively long N-terminal intracellular sequence, a single transmembrane region, an array of 8 EGF-like repeats that form cis-dimers via two disulfide bonds, Ig-like and b-propeller domains, a large b-barrel domain and a C-terminal toxin-like domain (see ‘Synaptic Latrophilin Complexes’ Figure)^1-3. The b-barrel and b-propeller domains and the C-terminal toxin-like domains of teneurins both originate evolutionarily from bacterial toxins but are independently derived in evolution from different bacteria³. Teneurins are encoded by four genes in mammals (Tenm1-4 in mice) that, like those of Lphns, are more broadly expressed during development but primarily transcribed in brain in adults. No domains similar to the b-barrel and b-propeller domains and the C-terminal toxin-like domains of teneurins are found in other mammalian proteins, rendering teneurins rather unusual mammalian proteins. Nearly all neurons express at least one teneurin gene, but most express multiple teneurin genes. Teneurins form constitutive cis-dimers but it is unknown if different teneurins heterodimerize.

Interactions. The b-barrel domain of teneurins binds with high affinity to the lectin-like domain of Lphns^4-6. In addition, two lines of evidence suggested that teneurin cis-dimers form non-covalent trans-dimers: the aggregation of cells expressing teneurins with each other^7,8, and the observation of such trans-dimers in crystal structures of teneurins^9,10. However, teneurins mediate cell aggregation via homophilic interactions only in one particular cell type and not in cell types normally used for cell-adhesion assays^11,12, the trans-dimers observed structurally are only observed in the absence of Lphn ligands^9,10 and could be packing artifacts, and only deletions of pre- but not of postsynaptic teneurins cause a synaptic phenotype¹³.

Functions. Presynaptic but not postsynaptic deletions of Tenm3 and Tenm4 in the hippocampal formation produces synapse loss, consistent with a heterophilic synaptic function mediated by binding to Lphns¹³. Constitutive KOs of various teneurins in mice cause a range of phenotypes, including in muscle and oligodendrocytes whose mechanisms remain unknown. Teneurins have also been implicated in axonal pathfinding but the evidence for such a function is indirect.

Knowledge gaps. The scope of teneurin functions is incompletely understood, as is their mechanism of action of teneurins not only in synapse formation, but also in all of their other apparent functions. Only a single high-affinity ligand, Lphn, was identified for teneurins that likely have additional ligands mediating diverse functions, given that these very large proteins have many more potential binding sites than the smaller FLRTs that interact with at least two ligands (see below). Moreover, it is unknown whether different teneurin and Lphn isoforms exhibit similar or distinct binding affinities that might account for synapse specificity. Although trans-homophilic interactions of teneurin dimers are proposed as a general specificity mechanism during circuit development, the affinities of various isoform interactions and their in vivo significance have not been tested. Furthermore, it is unknown what teneurin domains are functionally required for any of their roles. For example, is teneurin cis-dimerization via its EGF-like domains important, do the domains implicated in trans-homomultimerization have an essential function, and do the various toxin-like domains perform a critical role? Thus, as always much remains to be done.

References

1.     Araç D, Li J (2019) Teneurins and latrophilins: two giants meet at the synapse. Curr. Opin. Struct. Biol. 54, 141-151. 

2.     Jackson, V.A., Busby, J.N., Janssen, B.J.C., Lott, J.S., Seiradake, E. (2019) Teneurin Structures Are Composed of Ancient Bacterial Protein Domains. Front. Neurosci. 13, 183.

3.     Tucker RP (2018) Teneurins: Domain Architecture, Evolutionary Origins, and Patterns of Expression. Front. Neurosci. 12, 938.

4.     Li, J., Shalev-Benami, M., Sando, R., Jiang, X., Kibrom, A., Wang, J., Leon, K., Katanski, C., Nazarko, O., Lu, Y.C., Südhof, T.C., Skiniotis, G., and Araç, D. (2018) Structural basis for teneurin function in circuit-wiring: A toxin motif at the synapse. Cell 173, 735-748.

5.     Del Toro D, Carrasquero-Ordaz MA, Chu A, Ruff T, Shahin M, Jackson VA, Chavent M, Berbeira-Santana M, Seyit-Bremer G, Brignani S, Kaufmann R, Lowe E, Klein R, Seiradake E (2020) Structural Basis of Teneurin-Latrophilin Interaction in Repulsive Guidance of Migrating Neurons. Cell 180, 323-339.

6.     Li, J., Xie, Y., Cornelius, S., Jiang, X., Sando, R., Kordon, S., Pan, M., Leon, K., Südhof, T.C., Zhao, M., and Araç, D. (2020) Alternative splicing controls teneurin-latrophilin interaction and synapse specificity by a shape-shifting mechanism. Nature Comm. 11, 2140.

7.     Rubin, B.P., Tucker, R.P., Brown-Luedi, M., Martin, D., Chiquet-Ehrismann, R. (2002) Teneurin 2 is expressed by the neurons of the thalamofugal visual system in situ and promotes homophilic cell-cell adhesion in vitro. Development 129, 4697-4705.

8.     Berns, D.S., DeNardo, L.A., Pederick, D.T., Luo, L. (2018) Teneurin-3 controls topographic circuit assembly in the hippocampus. Nature 554, 328-333.

9.     Meijer, D.H., Frias, C.P., Beugelink, J.W., Deurloo, Y.N., Janssen, B.J.C. (2022) Teneurin4 dimer structures reveal a calcium-stabilized compact conformation supporting homomeric trans-interactions. EMBO J. 41, e107505.

10.  Li, J., Bandekar, S.J., Araç, D. (2023) The structure of fly Teneurin-m reveals an asymmetric self-assembly that allows expansion into zippers. EMBO Rep. 24, e56728.

11.  Boucard, A., Maxeiner, S., and Südhof, T.C. (2014) Latrophilins Function as Heterophilic Cell-Adhesion Molecules by Binding to Teneurins: Regulation by Alternative Splicing. J. Biol. Chem. 289, 387-402.

12.  Sando, R., Jiang, X., and Südhof, T.C. (2019) Latrophilin GPCRs direct synapse specificity by coincident binding of FLRTs and teneurins. Science 363, pii: eaav7969.

13.  Zhang, X., Lin, P.Y., Liakath-Ali, K, and Südhof, T.C. (2022) Teneurins Assemble into Presynaptic Nanoclusters that Promote Synapse Formation via Postsynaptic Non-Teneurin Ligands. Nature Comm. 13, 2297.

Lphn-FLRT Complexes

Properties. FLRTs comprise a family of three type-I membrane proteins (FLRT1-3) that are composed of an N-terminal leucine-rich repeat domain (10 leucine-rich repeats flanked by classical N- and C-terminal ‘cap’ sequences; see panel B in Figure above), a single fibronectin type III (FN3) domain, a transmembrane region, and a short non-conserved cytoplasmic sequence. FLRTs homodimerize via their leucine-rich repeat domains with a weak affinity in solution that may be enhanced on the membrane surface¹. Like Lphns and teneurins, FLRTs are more broadly expressed during development but are primarily synthesized in neurons in adults.

Interactions. FLRTs bind to the olfactomedin-like domain of Lphns on one side of the leucine-rich repeat domain and to the 1^st Ig domain of Unc5 on the other side^1,2. Binding of Lphn is compatible with homodimerization, whereas binding of Unc5 is not, although FLRTs can bind simultaneously as monomers to Lphns and to Unc5¹. Thus, it is possible that Lphn-teneurin complexes engage in interactions with FLRT dimers that expand into large mega-complexes containing FLRT and teneurin dimers, or in interactions with FLRT-Unc5 complexes that comprise two Unc5-FLRT-Lphn moieties each attached to a subunit of the teneurin homodimer. In addition to binding to Lphns and Unc5, FLRTs were reported to bind to FGF receptors via their FNIII domain³. However, the data are limited to indirect assays, making it unclear if a direct interaction exists.

Functions. The function of FLRTs as repulsive signals in neuronal cell migration and axon guidance is well established³. In addition, FLRTs have also been implicated via the Unc5 interactions in macrophage activation⁴. Their role in synapses, however, has only been indirectly shown by the demonstration that mutant, FLRT-binding incompetent, Lphn3 is unable to rescue the loss of synapses observed in Lphn3 KO neurons⁵.

Knowledge gaps. Nearly all key questions about FLRT function in synapses remain unaddressed. Do FLRTs perform an essential function at synapses, and if so, what exactly is that function? Does this function require Unc5 binding or dimerization? How do FLRTs transduce ligand-binding signals if at all – maybe by Unc5 binding? Do different FLRTs perform distinct functions? And does Unc5 have a synaptic function? Much remains to be done, especially in view of the diverse interactions of Unc5 with other proteins (e.g., netrins, glypicans) that in turn are also known to bind to yet another set of synaptic proteins (e.g., DCC/neogenin-1, LRRTM4).

References

1.     Lu, Y.C., Nazarko, O., Sando, R., Salzman, G., Südhof, T.C., and Araç, D. (2015) Crystal Structure of the FLRT3-LPHN3 complex reveal specific protein-protein interactions.  Structure 23, 1678-1691.

2.     Jackson, V.A., et al. (2016) Super-complexes of adhesion GPCRs and neural guidance receptors. Nat. Commun. 7, 11184. 

3.     Peregrina, C., Del Toro, D. (2020) FLRTing Neurons in Cortical Migration During Cerebral Cortex Development. Front. Cell Dev. Biol. 8, 578506. 

4.     Fang, Y., Ma, K., Huang, Y.M., Dang, Y., Liu, Z., Xu, Y., Zheng, X.L., Yang, X., Huo, Y., Dai, X. (2023) Fibronectin leucine-rich transmembrane protein 2 drives monocyte differentiation into macrophages via the UNC5B-Akt/mTOR axis. Front. Immunol. 14, 1162004.

5.     Sando, R., Jiang, X., and Südhof, T.C. (2019) Latrophilin GPCRs direct synapse specificity by coincident binding of FLRTs and teneurins. Science 363, pii: eaav7969.