Mechanical forces are an essential part of everyday life. Fundamental physiological processes such as breathing, heartbeat, and hearing all rely on the proper application and transmission of mechanical forces. Interestingly, mechanical inputs also influence biological processes beyond the limits of human perception-such as tissue regeneration, cell migration, and differentiation. At the cellular level, mechanical cues from the environment must be converted into biochemical signals that the cell can interpret. This task is carried out by specialized receptors known as mechanosensors, typically located on the plasma membrane. Well-known examples include integrins, which facilitate cell-extracellular matrix interactions, and ion channels, which open in response to membrane stretch, allowing ion exchange. A more recently recognized class of mechanosensors is the adhesion G protein-coupled receptors (aGPCRs).

Belonging to the GPCR superfamily-defined by the characteristic seven-transmembrane (7TM) domain-aGPCRs were long overlooked. However, recent breakthroughs in structural and biochemical characterization have identified a defining feature of these receptors: the GPCR-Autoproteolysis Inducing (GAIN) domain. This domain is essential for autoproteolytic cleavage, resulting in the receptor being expressed at the plasma membrane as a non-covalently bound heterodimer. Additionally, the GAIN domain harbors a tethered agonist, known as the Stachel peptide, which can strongly trigger signaling when released.

Recent efforts to understand aGPCR mechanotransduction have focused on either in vivo models or single-molecule force spectroscopy on purified extracellular domains. In contrast, in vitro studies have typically relied on qualitative force delivery methods combined with transcription-based reporters to assess receptor activity. As a result, there remains a significant gap in in vitro platforms that can both quantitatively apply mechanical force and capture receptor activation through a direct, real-time readout, as opposed to relying on indirect transcriptional reporters.

In this thesis, we investigate GPR56/ADGRG1, a known shear stress sensor. Using a spinning disc device to deliver defined levels of fluid shear stress and a genetically encoded BRET biosensor to monitor G protein dissociation, we quantify receptor activation in HEK293T cells. Additionally, we apply confocal microscopy and computational image analysis to assess the extent of extracellular domain removal (shedding) following mechanical stimulation. Importantly, this setup is also used to study the mechanotransduction properties of previously computationally designed ADGRG1 GAIN domain mutants, establishing a foundation for mechano-design approaches with potential applications in synthetic biology.