Structural analysis and conformational dynamics of a holo-adhesion GPCR reveal interplay between extracellular and transmembrane domains

Abstract

Adhesion G Protein-Coupled Receptors (aGPCRs) are key cell-adhesion molecules involved in numerous physiological functions. aGPCRs have large multi-domain extracellular regions (ECR) containing a conserved GAIN domain that precedes their seven-pass transmembrane domain (7TM). Ligand binding and mechanical force applied on the ECR regulate receptor function. However, how the ECR communicates with the 7TM remains elusive, because the relative orientation and dynamics of the ECR and 7TM within a holoreceptor is unclear. Here, we describe the cryo-EM reconstruction of an aGPCR, Latrophilin3/ADGRL3, and reveal that the GAIN domain adopts a parallel orientation to the membrane and has constrained movement. Single-molecule FRET experiments unveil three slow-exchanging FRET states of the ECR relative to the 7TM within the holoreceptor. GAIN-targeted antibodies, and cancer-associated mutations at the GAIN-7TM interface, alter FRET states, cryo-EM conformations, and receptor signaling. Altogether, this data demonstrates conformational and functional coupling between the ECR and 7TM, suggesting an ECR-mediated mechanism of aGPCR activation.

The far extracellular CUB domain of the adhesion GPCR ADGRG6/GPR126 is a key regulator of receptor signaling

“…Li et al ., 2020) to instruct synapse specificity, and anti-lectin synthetic antibodies (sABs) can modulate signaling (Kordon et al ., 2023). Furthermore, for ADGRL3, anti-GAIN sABs which alter the GAIN/7TM relative orientation can affect receptor signaling (Kordon et al ., 2024), supporting an allosteric connection between ECR and 7TM in aGPCRs. ADGRG1 directs myelination through the ligand of its N-terminal PLL domain, transglutaminase-2 (Giera et al ., 2018; Salzman et al ., 2020), and anti-PLL nanobodies can alter signaling (Salzman et al ., 2017).…”

“…According to the tethered agonist (TA) model of aGPCR activation, removal of the ECR from the membrane-bound seven-pass transmembrane (7TM) region reveals a TA peptide which then activates the receptor (Araç et al ., 2012; Lala and Hall, 2022). A large pool of evidence shows that the ECR can also directly modulate 7TM signaling in a TA-independent manner (Kishore et al ., 2016; Salzman et al ., 2017; Leon et al ., 2020; Lala and Hall, 2022; Bui et al ., 2023; Bandekar et al ., 2024; Kordon et al ., 2024). In this second model, communication between ECR and the 7TM is important for the regulation of receptor activity (Lala and Hall, 2022; Kordon et al ., 2024).…”

“…A large pool of evidence shows that the ECR can also directly modulate 7TM signaling in a TA-independent manner (Kishore et al ., 2016; Salzman et al ., 2017; Leon et al ., 2020; Lala and Hall, 2022; Bui et al ., 2023; Bandekar et al ., 2024; Kordon et al ., 2024). In this second model, communication between ECR and the 7TM is important for the regulation of receptor activity (Lala and Hall, 2022; Kordon et al ., 2024). ECR conformation can be affected through the binding of a ligand or by alternative splicing (Araç and Leon, 2019; Leon et al ., 2020) and these events are communicated to the 7TM to alter signaling (Kordon et al ., 2024).…”

“…In this second model, communication between ECR and the 7TM is important for the regulation of receptor activity (Lala and Hall, 2022; Kordon et al ., 2024). ECR conformation can be affected through the binding of a ligand or by alternative splicing (Araç and Leon, 2019; Leon et al ., 2020) and these events are communicated to the 7TM to alter signaling (Kordon et al ., 2024). In further support of this idea, roughly half of aGPCRs lack TA-dependent signaling (Dates et al ., 2024).…”