A single domain intrabody biases the follicle-stimulating hormone receptor subcellular trafficking

Variable fragments from camelid heavy-chain only antibodies (VHH) can stabilize active conformations of G protein-coupled receptors (GPCRs), enabling 3D structural characterization and supporting detailed structure–activity studies by linking receptor conformational states to downstream signaling. When expressed intracellularly, these VHHs have recently proven useful for tracking active GPCRs across subcellular compartments. Here, we report the isolation and characterization of iPRC2, an intra-VHH that selectively recognizes intracellular loops of the human follicle-stimulating hormone receptor (hFSHR). Expression of iPRC2 in hFSHR-expressing cells reduces FSH-induced cAMP accumulation, while requiring G α s for optimal receptor interaction. Importantly, iPRC2 promotes hFSHR accumulation in early endosomes and limits its recycling to the cell surface. Hence, unlike previously described intra-VHHs that recognize active GPCR intracellular location, iPRC2 induces per se a location bias. By reshaping hFSHR trafficking, iPRC2 provides a unique live-cell tool to manipulate receptor localization and investigate how compartmentalized GPCR trafficking influences signaling outputs.