Convergent selective signaling impairment exposes the pathogenicity of latrophilin-3 missense variants linked to inheritable ADHD susceptibility

Abstract

Latrophilin-3 (Lphn3; also known as ADGRL3) is a member of the adhesion G Protein Coupled Receptor subfamily, which participates in the stabilization and maintenance of neuronal networks by mediating intercellular adhesion through heterophilic interactions with transmembrane ligands. Polymorphisms modifying the Lphn3 gene are associated with attention-deficit/hyperactivity disorder (ADHD) in children and its persistence into adulthood. How these genetic alterations affect receptor function remains unknown. Here, we conducted the functional validation of distinct ADHD-related Lphn3 variants bearing mutations in the receptor’s adhesion motif-containing extracellular region. We found that all variants tested disrupted the ability of Lphn3 to stabilize intercellular adhesion in a manner that was distinct between ligands classes, but which did not depend on ligand-receptor interaction parameters, thus pointing to altered intrinsic receptor signaling properties. Using G protein signaling biosensors, we determined that Lphn3 couples to Gαi1, Gαi2, Gαs, Gαq, and Gα13. However, all ADHD-related receptor variants consistently lacked intrinsic as well as ligand-dependent Gα13 coupling efficiency while maintaining unaltered coupling to Gαi, Gαs, and Gαq. Consistent with these alterations, actin remodeling functions as well as actin-relevant RhoA signaling normally displayed by the constitutively active Lphn3 receptor were impeded by select receptor variants, thus supporting additional signaling defects. Taken together, our data point to Gα13 selective signaling impairments as representing a disease-relevant pathogenicity pathway that can be inherited through Lphn3 gene polymorphisms. This study highlights the intricate interplay between Lphn3 GPCR functions and the actin cytoskeleton in modulating neurodevelopmental cues related to ADHD etiology.

Thwarting of Lphn3 Functions in Cell Motility and Signaling by Cancer-Related GAIN Domain Somatic Mutations

“…Given that Lphn3 adhesion functions are mediated through heterophilic ligand-receptor interactions stabilized by its extracellular adhesion domains, we focused on cancer-related mutations specifically located in Lphn3 extracellular GAIN domain ( Figure 1 a) to investigate their impact on the receptor’s intercellular adhesive properties involving endogenous transmembrane ligands, Flrt3 and Teneurin2. Although Flrt3 and Teneurin2 interact with Lphn3 olfactomedin-like and lectin-like domains, respectively, extracellular mutations have been reported to impair Lphn3-mediated cell-cell adhesion despite being located outside of the receptor-ligand interface [ 30 ]. In order to assess the impact of these mutations on Lphn3-mediated heterotypic cell adhesion profile, we monitored the formation of cell-cell contacts through the implementation of cell aggregation assays, using heterologous expression in HEK293 cells, and which consisted of the mixing of Lphn3 variants-expressing cells with Flrt3/Teneurin2-expressing cells.…”

“…Taken together, these results point to a description of Lphn3-mediated intercellular adhesion that does not strictly depend on receptor-ligand interactions, but might involve additional factors, which preferentially destabilize Flrt3- and not Teneurin2-dependent adhesion complexes. Such factors are likely to originate from the receptors’ intrinsic conformational states thought to allow the conversion of mechanical/adhesive stimuli into the activation of intracellular signaling pathways, a feature attributed to its CTF rather than its NTF, and previously described to explain constitutive, as well as ligand-dependent activity of Lphn3 [ 30 , 35 , 36 ]. The ability of Lphn3 to signal through both G protein-dependent and -independent pathways towards the actin cytoskeleton raises the possibility that its adhesive units might rely on cytoskeletal anchoring to sustain tensile forces, such as the ones detected in aggregation assays, during which shear forces are generated.…”

“…An inherent property of the Lphn3 receptor resides in its ability to activate cell-autonomous intracellular signaling cascades in a ligand-independent manner notably by coupling to various families of G proteins, but preferably to the G13 family subset [ 30 ]. Lphn3 GAIN domain is known to encrypt a tethered ligand that can interact with the CTF’s transmembrane segments through various degrees of exposure made possible by dynamic rearrangements of the GAIN domain itself [ 37 ].…”

“…This oncogenic role was corroborated by the detected overexpression of Lphn3 in cancer cell lines originating from different tissues such as prostate, lung, breast and leukocytes [ 27 ]. While it is unknown which signaling pathways would involve Lphn3 functions in cancerous processes, its ability to couple with G13 protein and its actin cytoskeleton remodeling activities are among the functions that are susceptible to being targeted [ 28 , 29 , 30 ]. Given the potential role of the GAIN domain in aGPCR activation, we sought to investigate the functional defects induced by the presence of single amino acid substitutions into Lphn3 GAIN domain originating from genetic somatic mutations identified in lung cancer primary tumors [ 25 ].…”

“…The following supporting information can be downloaded at: , Figure S1: Flrt3 binding is restricted to cells expressing Lphn3 or its variants; Figure S2: Extended data for immunoblot images; Figure S3: Vimentin rearrangement in Lphn3-expressing cells coincides with nuclear deformations; Figure S4: Flrt3 does not act as a migration-inducing agent in wound healing assays but expression of Lphn3 or cancer-associated GAIN variants cell-autonomously reduces the migration potential of HEK293 cells; Video S1: Time-lapse fluorescence confocal microscopy imaging of HEK293 cells expressing mVenus-tagged Lphn3-WT stimulated with Flrt3; Video S2: Time-lapse fluorescence confocal microscopy imaging of HEK293 cells expressing mVenus; Video S3: Time-lapse confocal microscopy fluorescence imaging of HEK293 cells expressing mVenus-tagged Lphn3-WT; Video S4: Time-lapse confocal microscopy fluorescence imaging of HEK293 cells expressing mVenus-tagged S810L mutant receptor. References [ 29 , 30 , 31 , 61 , 62 , 63 , 64 , 65 ] are cited in Supplementary Materials.…”