Control of G protein-coupled receptor function via membrane-interacting intrinsically disordered C-terminal domains.

G protein-coupled receptors (GPCRs) control intracellular signaling cascades via agonist-dependent coupling to intracellular transducers including heterotrimeric G proteins, GPCR kinases (GRKs), and arrestins. In addition to their critical interactions with the transmembrane core of active GPCRs, all three classes of transducers have also been reported to interact with receptor C-terminal domains (CTDs). An underexplored aspect of GPCR CTDs is their possible role as lipid sensors given their proximity to the membrane. CTD-membrane interactions have the potential to control the accessibility of key regulatory CTD residues to downstream effectors and transducers. Here, we report that the CTDs of two closely related family C GPCRs, metabotropic glutamate receptor 2 (mGluR2) and mGluR3, bind to membranes and that this interaction can regulate receptor function. We first characterize CTD structure with NMR spectroscopy, revealing lipid composition-dependent modes of membrane binding. Using molecular dynamics simulations and structure-guided mutagenesis, we then identify key conserved residues and cancer-associated mutations that modulate CTD-membrane binding. Finally, we provide evidence that mGluR3 transducer coupling is controlled by CTD-membrane interactions in live cells, which may be subject to regulation by CTD phosphorylation and changes in membrane composition. This work reveals an additional mechanism of GPCR modulation, suggesting that CTD-membrane binding may be a general regulatory mode throughout the broad GPCR superfamily.

Kartogenin-loaded coaxial PGS/PCL aligned nanofibers for cartilage tissue engineering.

Electrospinning is a valuable technology for cartilage tissue engineering (CTE) due to its ability to produce fibrous scaffolds mimicking the nanoscale and alignment of collagen fibers present within the superficial zone of articular cartilage. Coaxial electrospinning allows the fabrication of core-shell fibers able to incorporate and release bioactive molecules (e.g., drugs or growth factors) in a controlled manner. Herein, we used coaxial electrospinning to produce coaxial poly(glycerol sebacate) (PGS)/poly(caprolactone) (PCL) aligned nanofibers (core:PGS/shell:PCL). The obtained scaffolds were characterized in terms of their structure, chemical composition, thermal properties, mechanical performance and in vitro degradation kinetics, in comparison to monoaxial PCL aligned fibers and respective non-aligned controls. All the electrospun scaffolds produced presented average fiber diameters within the nanometer-scale and the core-shell structure of the composite fibers was clearly confirmed by TEM. Additionally, fiber alignment significantly increased (>2-fold) the elastic modulus of both coaxial and monoxial scaffolds. Kartogenin (KGN), a small molecule known to promote mesenchymal stem/stromal cells (MSC) chondrogenesis, was loaded into the core PGS solution to generate coaxial PGS-KGN/PCL nanofibers. The KGN release kinetics and scaffold biological performance were evaluated in comparison to KGN-loaded monoaxial fibers and respective non-loaded controls. Coaxial PGS-KGN/PCL nanofibers showed a more controlled and sustained KGN release over 21 days than monoaxial PCL-KGN nanofibers. When cultured with human bone marrow MSC in incomplete chondrogenic medium (without TGF-ß3), KGN-loaded scaffolds enhanced significantly cell proliferation and chondrogenic differentiation, as suggested by the increased sGAG amounts and chondrogenic markers gene expression levels. Overall, these findings highlight the potential of using coaxial PGS-KGN/PCL aligned nanofibers as a bioactive scaffold for CTE applications.