A Membrane-proximal, C-terminal α-Helix Is Required for Plasma Membrane Localization and Function of the G Protein-coupled Receptor (GPCR) TGR5

Background: TGR5 is a G protein-coupled bile acid receptor that modulates the immune response, glucose homeostasis, and liver regeneration. Results: Secondary structure of the receptor C terminus determines plasma membrane trafficking. Conclusion: TGR5 plasma membrane content and responsiveness to extracellular ligands depends on C-terminal -helix formation. Significance: This provides insights into the structure-function relationship of TGR5, which is a potential drug target for metabolic diseases. The C terminus of G protein-coupled receptors (GPCRs) is important for G protein-coupling and activation; in addition, sorting motifs have been identified in the C termini of several GPCRs that facilitate correct trafficking from the endoplasmic reticulum to the plasma membrane. The C terminus of the GPCR TGR5 lacks any known sorting motif such that other factors must determine its trafficking. Here, we investigate deletion and substitution variants of the membrane-proximal C terminus of TGR5 with respect to plasma membrane localization and function using immunofluorescence staining, flow cytometry, and luciferase assays. Peptides of the membrane-proximal C-terminal variants are subjected to molecular dynamics simulations and analyzed with respect to their secondary structure. Our results reveal that TGR5 plasma membrane localization and responsiveness to extracellular ligands is fostered by a long ( 9 residues) -helical stretch at the C terminus, whereas the presence of -strands or only a short -helical stretch leads to retention in the endoplasmic reticulum and a loss of function. As a proof-of-principle, chimeras of TGR5 containing the membrane-proximal amino acids of the (2) adrenergic receptor ((2)AR), the sphingosine 1-phosphate receptor-1 (S1P1), or the -type opioid receptor (OR) were generated. These TGR5(2)AR, TGR5S1P1, or TGR5OR chimeras were correctly sorted to the plasma membrane. As the exchanged amino acids of the (2)AR, the S1P1, or the OR form -helices in crystal structures but lack significant sequence identity to the respective TGR5 sequence, we conclude that the secondary structure of the TGR5 membrane-proximal C terminus is the determining factor for plasma membrane localization and responsiveness towards extracellular ligands.