Single Piconewton Forces Regulate Dissociation of the Latrophilin-3 Gain Domain

Latrophilins are adhesion G-protein coupled receptors (aGPCRs) that have been implicated in multiple neuropsychiatric diseases and are necessary for excitatory synapse formation in specific regions of the brain. Despite the importance of latrophilins in neurodevelopment, the mechanisms involved in signaling by latrophilins, and aGPCRs in general, remain poorly understood. Latrophilins are autoproteolytically cleaved at a GAIN domain conserved across aGPCRs, and the two resulting latrophilin fragments remain stably (but non-covalently) associated on the cell membrane. In one prevalent hypothesis, dissociation of the latrophilin fragments, potentially in response to mechanical forces applied through transsynaptic ligand binding, drives a conformational change in the transmembrane domain to initiate signal transduction. However, the stability of the association between latrophilin fragments calls into question how and whether their dissociation can occur on biologically relevant timescales. Here, we present evidence that physiological levels of mechanical force substantially accelerate the rate of dissociation of the Latrophilin-3 (Lphn3) GAIN domain. Using magnetic tweezers, we directly apply mechanical force in the single-pN range to individual Lphn3 GAIN domains and demonstrate that these forces are sufficient to dissociate the GAIN domain on the seconds-to-minutes timescale. We further observe that the GAIN domain fragments can reversibly reassociate after force-mediated dissociation. Moreover, the distribution of measured Lphn3 dissociation times suggests that the fragments of the Lphn3 GAIN domain form a two-state slip bond, with a highly force-sensitive transition between strongly- and weakly-bound states. Our results support the idea that mechanical force may be a key regulator of latrophilin signaling in synapse formation and, more broadly, signaling by aGPCRs.