The role of Gαs in G protein-coupled receptor (GPCR) signalling at the cell surface is well established. Recent evidence has revealed the presence of Gαs on endosomes and its capacity to elicit GPCR-promoted signalling from this intracellular compartment. Here, we report an unconventional role for Gαs in the endocytic sorting of GPCRs to lysosomes. Cellular depletion of Gαs specifically delays the lysosomal degradation of GPCRs by disrupting the transfer of GPCRs into the intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). We show that Gαs interacts with GASP-1 and dysbindin, two key proteins that serve as linkers between GPCRs and the ESCRT (endosomal sorting complex required for transport) machinery involved in receptor sorting into ILVs. Our findings reveal that Gαs plays a role in both GPCR signalling and trafficking pathways, providing another piece in the intertwining molecular network between these processes.

IntroductionThe microenvironment surrounding inflamed synovium leads to the activation of fibroblast-like synoviocytes (FLSs), which are important contributors to cartilage destruction in rheumatoid arthritic (RA) joints. Transglutaminase 2 (TG2), an enzyme involved in extracellular matrix (ECM) cross-linking and remodeling, is activated by inflammatory signals. This study was undertaken to assess the potential contribution of TG2 to FLS-induced cartilage degradation.MethodsTransglutaminase (TGase) activity and collagen degradation were assessed with the immunohistochemistry of control, collagen-induced arthritic (CIA) or TG2 knockdown (shRNA)-treated joint tissues. TGase activity in control (C-FLS) and arthritic (A-FLS) rat FLSs was measured by in situ 5-(biotinamido)-pentylamine incorporation. Invadopodia formation and functions were measured in rat FLSs and cells from normal (control; C-FLS) and RA patients (RA-FLS) by in situ ECM degradation. Immunoblotting, enzyme-linked immunosorbent assay (ELISA), and p3TP-Lux reporter assays were used to assess transforming growth factor-β (TGF-β) production and activation.ResultsTG2 and TGase activity were associated with cartilage degradation in CIA joints. In contrast, TGase activity and cartilage degradation were reduced in joints by TG2 knockdown. A-FLSs displayed higher TGase activity and TG2 expression in ECM than did C-FLSs. TG2 knockdown or TGase inhibition resulted in reduced invadopodia formation in rat and human arthritic FLSs. In contrast, increased invadopodia formation was noted in response to TGase activity induced by TGF-β, dithiothreitol (DTT), or TG2 overexpression. TG2-induced increases in invadopodia formation were blocked by TGF-β neutralization or inhibition of TGF-βR1.ConclusionsTG2, through its TGase activity, is required for ECM degradation in arthritic FLS and CIA joints. Our findings provide a potential target to prevent cartilage degradation in RA.