Biology subjects

Langenhan, T.

Publications and source records attributed to Langenhan, T..

9 recordsLinked to original sources

bioRxiv2025

Dual-mode intramolecular agonist-dependent mechanoactivation of the adhesion GPCR ADGRG1/GPR56

Mechanical stimuli instruct cardinal cellular decisions pertaining to cell fate, proliferation, morphology, and movement1,2. How adhesion G protein-coupled receptors (aGPCRs), a large family of mechanosensors with more than 30 members in humans3-5, transduce mechanical cues into metabotropic commands has been a matter of debate due to the lack of suitable approaches to analyze receptor activation during mechanotransduction in live cells6. A central question is whether aGPCR activation strictly requires mechanical dissociation of the autoproteolyzed N- and C-terminal fragment (NTF and CTF) complex to expose the tethered intramolecular agonist (TIA; 'Stachel'). Here, we investigated human ADGRG1/GPR56 (G1), an aGPCR with roles in brain development7, skeletal muscle8, and platelet function9, to study the events during aGPCR mechanotransduction by monitoring receptor dissociation and G protein recruitment in real time during cell migration and substrate deformation. Magnetic-tweezer measurements at a physiologically relevant loading rate showed that isolated G1 GAIN domains can partially unfold and refold at forces below those required for NTF-CTF dissociation. In cells, G1 dissociation occurred at retracting fibers during cell migration10 over an adhesive substrate through its native extracellular matrix ligand, tissue transglutaminase 2 (TG2), and was enhanced by cell stretching. Simultaneous live recording of G protein recruitment and pharmacological assays during mechanical stimulation of G1 reveal a dual-mode activation mechanism: mechanical force elicits robust metabotropic signaling without requiring NTF dissociation and generates an intracellular response commensurate with the fully dissociated receptor. Both signaling modes depend on an intact Stachel element11,12 of the receptor under our experimental conditions. Our findings determine mechanotransduction principles governing aGPCR activation at the cell-ECM interface. We reveal that G1 does not function as a simple binary switch, but rather as a highly sophisticated mechanosensor that dynamically encodes localized extracellular tension into distinct, highly scalable metabotropic outputs.

cell biology↗

bioRxiv2025

Polycystin-1 acts as an atypical adhesion GPCR that responds to novel Wnt signaling and mechanical stimuli

Mutations in the genes encoding Polycystin-1 and 2 cause autosomal dominant polycystic kidney disease (ADPKD). These proteins cellular functions and the mechanisms through which their absence causes disease are unknown. We find that the behavior of the 11 transmembrane domain polycystin-1 protein resembles that of an adhesion G protein-coupled receptor when activated by Wnt ligands. Exposure to Wnt ligands causes shedding of the polycystin-1 N terminal region, exposing a tethered agonist that activates G protein-dependent functions of the membrane-associated C terminal fragment. Activated polycystin-1 communicates through G13 to activate RhoA, leading in turn to the ROCK-dependent phosphorylation and inactivation of GSK3{beta}. Activated polycystin-1 traffics out of the primary cilium through an arrestin-dependent mechanism, and arrestin involvement is required for signaling to occur. These data elaborate a function for polycystin-1 that is structurally non-canonical and that is directly connected to pathways whose perturbation results in cystic disease. SummaryThe polycystin-1 protein can activate a signaling pathway resembling those employed by non-canonical adhesion G protein coupled receptors, which is induced by Wnt9b binding and which leads to leads to inhibition of the GSK3{beta} kinase and removal of polycystin-1 from the primary cilium. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/655326v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@1b32dc1org.highwire.dtl.DTLVardef@80ee43org.highwire.dtl.DTLVardef@9aa441org.highwire.dtl.DTLVardef@95ceb1_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract PC1 resides at baseline in the primary cilium. PC1 behaves as an aGPCR that responds to Wnt9b and to flow-induced ATP release by departing from the cilium and initiating the inhibition of GSK3. The schematic diagram presents a model that integrates the findings presented here. Wnt9b binding to the PC1 NTF leads to shedding of the NTF, exposing the tethered agonist to activate the PC1 receptor-mediated signaling cascade, which involves triggering G13 binding and the subsequent nucleotide exchange and GTP-loading of RhoA. GTP-bound RhoA stimulates the kinase activity of ROCK, which phosphorylates and inhibits GSK3. C_FIG

cell biology↗

bioRxiv2023

Drosophila Bchs overexpression recapitulates human WDFY3 neurodevelopmental phenotypes with implications for glial cell involvement in altered head circumference

The autophagy adaptor WDFY3 is linked to neurodevelopmental delay and altered brain size. Loss-of-function variants are associated with an increased brain size in both humans and mice. We thus, hypothesized that the microcephaly observed in some of the patients may be related to a gain-of-function of the WDFY3 gene product. While the role of WDFY3 loss-of-function has been studied extensively in neurons, little is known about the effects of WDFY3 overexpression in different neural cell types. We utilized a Drosophila melanogaster overexpression model to investigate the effect of the WDFY3 ortholog Bchs (blue cheese) on development, CNS size, and gene expression profiles. Glial and neuronal overexpression of Bchs impaired CNS development, locomotion and autophagy. Glial overexpression of Bchs also altered CNS size significantly. We identified 79 genes that were differentially expressed and overlapped in flies that overexpress Bchs in glial and neuronal cells, respectively. Additionally, upon neuronal Bchs overexpression differentially expressed genes clustered in gene ontology categories associated with autophagy and mitochondria. Our data indicate that WDFY3/Bchs overexpression in both neurons and glial cells results in impaired neural development, which corresponds to symptoms observed in WDFY3-related neurodevelopmental delay.

neuroscience↗

bioRxiv2023

Neuronal correlates of time integration into memories

The circadian clock affects a wide range of physiological processes. Of particular interest is the influence of the clock on memory performance, as circadian dysfunction is associated with age- and disease-related decline in memory. In various species it has been shown that memory performance is regulated by the circadian clock. However, the anatomical and functional connection of the circadian clock and memory neurons has not been described in detail so far. This study now identifies that Diuretic hormone 31 (DH31)-positive clock neurons of the DN1p cluster regulate memory performance. DH31, a functional homolog of the mammalian calcitonin gene-related peptide, plays a crucial role in this process as a clock communication signal. DH31 facilitates memory performance during the night via indirect signalling, while DH31 signals directly to the mushroom bodies restricting memory performance specifically in the evening. This pleiotropic action of DH31 suggests that the circadian clock confines memory performance to a physiological dynamic range.

neuroscience↗

bioRxiv2023

Step-wise mechanical unfolding and dissociation of the GAIN domains of ADGRG1/GPR56, ADGRL1/Latrophilin-1 and ADGRB3/BAI3: insights into the mechanical activation hypothesis of adhesion G protein-coupled receptors

Adhesion G protein-coupled receptors (aGPCRs) are a large family within the superfamily of G protein-coupled receptors involved in various physiological processes. One unique feature of aGPCRs is their long N-terminal extracellular regions (ECRs), which contain adhesive domains and a GPCR autoproteolysis-inducing (GAIN) domain. This GAIN domain promotes autoproteolytic cleavage of aGPCRs into N- and C-terminal fragments (NTF, CTF, respectively) after receptor biosynthesis. aGPCR signaling involves an interplay between the NTF and CTF that can be me-chanically activated or modulated. However, how force affects the conformation/structure of the GAIN domain as a central structural element in aGPCR activation remains largely unknown. In this study, we investigated the mechanical stability of the GAIN domains of three aGPCRs from subfamilies B, G and L at a loading rate of 1 pN/s. Our findings demonstrate that the GAIN domains can be destabilized by forces from a few to 20 piconewtons (pN). Specifically, for the autocleaved aGPCRs, ADGRG1/GPR56 and ADGRL1/Latrophilin-1, forces over this range can cause detachment of the GAIN domain from the membrane-proximal Stachel element, which serves as an endogenous tethered agonist to aGPCRs, typically preceded with GAIN domain unfolding. For the non-cleavable aGPCR ADGRB3/BAI3, the GAIN domain undergoes complex mechanical unfolding over a similar force range. We also demonstrate that detachment of the GAIN domain can take place during cell migration, provided that the linkage between aGPCR and extracellular matrix is sufficiently stable. These results suggest that both structural stability of the GAIN domain and NTF/CTF dissociation are sensitive to physiological ranges of tensile forces, providing insights into the mechanical activation hypothesis of aGPCRs.

biophysics↗

bioRxiv2023

Structural basis of GAIN domain autoproteolysis and cleavage-resistance in the adhesion G-protein coupled receptors

The GAIN domain is a hallmark of adhesion G-protein coupled receptors (aGPCRs) as this extracellular domain contains an integral agonistic sequence (Stachel) for activation via binding to the 7-transmembrane helical (7TM) domain of the receptor. Many aGPCRs are autoproteolytically cleaved at the GPCR proteolysis site (GPS) site within the GAIN domain formed HXS/T sequence motif. However, other aGPCR can be activated without GPS cleavage. We determined the crystal structure of the human ADGRB2/BAI2 hormone receptor (HormR) and GPCR autoproteolysis-inducing (GAIN) domains and found that this aGPCR is resistant to autoproteolysis despite the presence of a canonical HLS sequence motif at the GPS. We used structural comparisons and molecular dynamics (MD) simulations to identify structural determinants that are important for autocleavage beyond the canonical HXS/T motif. These studies characterized a conserved glycine residue and an edge-{pi} interaction of the histidine base of the GPS sequence with a phenylalanine residue that is highly conserved in cleavage-competent aGPCRs. The MD simulations showed that this interaction is important to position the imidazole group of the histidine for deprotonation of the serine or threonine nucleophile. Removal of this interaction reduced autoprote-olytic activity in the ADGRL1 receptor and restored cleavage competence of the ADGRB3 receptor in a R866H/L821F double mutant. Conservation analysis indicates that wild-type ADGRB2 and ADGRB3 are auto-cleavage-incompetent receptors.

biochemistry↗

bioRxiv2023

Intron retention of an adhesion GPCR generates single transmembrane-helix isoforms to enable 7TM-adhesion GPCR function

Adhesion G protein-coupled receptors (aGPCR) function as metabotropic mechanosensors in the nervous system and other organs. aGPCR are heavily spliced forecasting an extraordinary molecular structural diversity. Many predicted isoforms lack the transmembrane (7TM) signaling subunit, but to what extent these non-GPCR isoforms are produced and what physiological purpose they serve is unknown. Alternative splicing through intron retention of ADGRL/Latrophilin/Cirl mRNA in Drosophila generates transcripts encoding unconventional proteins with an extracellular domain anchored by a single transmembrane helix (Cirl1TM). Here, we show that Cirl1TM transcripts are translated in vivo and that Cirl1TM binds Cirl7TM N-terminal fragment-dependently. This interaction enables mechanosensory neurons to distinguish input intensities through Go-dependent signaling. Similarly, a direct interaction was found for mammalian GPR126/ADGRG6 isoforms. Together, our findings define intron retention and isoform-specific heteromerization as extraordinary molecular strategies to adjust Cirl-dependent mechanosensation and demonstrate physiological relevance of versatile aGPCR isoform repertoire to tune cellular responsiveness.

neuroscience↗

bioRxiv2020

Antinociceptive modulation by the adhesion-GPCR CIRL promotes mechanosensory signal discrimination

Adhesion-type G protein-coupled receptors (aGPCRs) participate in a vast range of physiological processes. Correspondingly, these receptors are associated with diverse human diseases, such as developmental disorders, defects of the nervous system, allergies and cancer. Several aGPCRs have recently been linked to mechanosensitive functions suggesting that processing of mechanical stimuli may be a common feature of this receptor family. CIRL (ADGRL/Latrophilin, LPHN), one of the oldest members of the aGPCR family, sensitizes sensory responses of larval Drosophila to gentle touch and sound by amplifying mechanosensory signal transduction in low-threshold mechanoreceptors (Scholz et al., 2015; 2017). In the present study, we show that Cirl is also expressed in high-threshold mechanical nociceptors where it adjusts nocifensive behaviour under physiological and pathophysiological conditions. Optogenetic in vivo experiments indicate that CIRL quenches cAMP levels in both mechanosensory submodalities. However, contrasting its effect in touch sensitive neurons, CIRL dampens the response of nociceptors to mechanical stimulation. Consistent with this finding, rat nociceptors display a drop in Cirl1 expression during allodynia. Taken together, these results demonstrate that CIRL exerts opposing modulatory functions in low-threshold mechanosensors and high-threshold nociceptors. This intriguing bipolar action likely facilitates the separation of mechanosensory signals carrying different physiological information.

physiology↗

bioRxiv2018

Complexin cooperates with Bruchpilot to tether synaptic vesicles to the active zone cytomatrix

Information processing by the nervous system depends on the release of neurotransmitter from synaptic vesicles (SVs) at the presynaptic active zone. Molecular components of the cytomatrix at the active zone (CAZ) regulate the final stages of the SV cycle preceding exocytosis and thereby shape the efficacy and plasticity of synaptic transmission. Part of this regulation is reflected by a physical association of SVs with filamentous CAZ structures. However, our understanding of the protein interactions underlying SV tethering by the CAZ is far from complete. The very C-terminal region of Bruchpilot (Brp), a key component of the Drosophila CAZ, participates in SV tethering. Yet so far, no vesicular or cytoplasmic molecules have been reported to engage in an interaction with Brps C-terminus. Here, we carried out an in vivo screen for molecules that link the Brp C-terminus to SVs. This strategy identified the conserved SNARE (soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor) regulator Complexin (Cpx) as a vesicular interaction partner of Brp. We show that Brp and Cpx interact genetically and functionally. Interfering with Cpx targeting to SVs mirrored distinctive features of a C-terminal Brp truncation: impaired SV recruitment to the CAZ and enhanced short-term synaptic depression. Extending the study beyond Drosophila synapses, we interrogated active zones of mouse rod bipolar cells. Here, too, we collected evidence for an evolutionarily conserved role of Cpx upstream of SNARE complex assembly where it participates in SV tethering to the CAZ.

neuroscience↗