A proximal α-helix of the TGR5 C-Terminus is essential for receptor plasma membrane localization

Introduction:

TGR5 is a G-protein coupled bile acid receptor (GPCR), which has been linked to bile acid and glucose homeostasis, energy expenditure and prevention of atherosclerosis. Thus, TGR5 represents an interesting drug target for liver and metabolic diseases. The crystal structure of TGR5 is unknown. We have previously demonstrated that the deletion of the 35 C-terminal amino acids (aa) results in endoplasmic reticulum (ER) retention of the mutated protein (1). The C-terminus of GPCRs is important for G protein-coupling and activation; in addition, the C-termini of several GPCRs are required for correct trafficking from the ER to the plasma membrane. Aim of the present study was to characterize the role of the TGR5 C-terminus for receptor localization.

Methods:

TGR5 mutations were introduced by site-directed-mutagenesis and cloning techniques. Impact on receptor localization was determined by immunofluorescence microscopy and FACS analysis. TGR5 function was studied using a cAMP sensitive luciferase reporter gene (1).

Results:

Truncation of the TGR5 C-terminus at aa 284 to 297 resulted in an accumulation of the mutated proteins in the ER and loss of function. To elucidate the role of the proximal TGR5 C-terminus deletion variants (Δ285 – 290; Δ291 – 297) as well as alanine, glycine or proline substitutions (285 – 290A/G/P; 291 – 297A/G/P) were generated. Since no known sorting signal was identified in the proximal C-terminus, these aa are most likely essential for correct folding of the protein and seem to be relevant for a transport-competent state. Computational analysis indicates that the proximal TGR5 C-terminus contains an α-helix, which is disrupted by the 285 – 290/291 – 297 variants thereby causing ER retention. As a proof-of-principle, a chimera of TGR5 containing the membrane-proximal amino acids of the β2 adrenergic receptor was generated that was correctly sorted to the plasma membrane and showed a similar functional activity as the TGR5 WT when stimulated with the bile acid agonist taurolithocholic acid (10µM).

Discussion/Conclusion:

The plasma membrane localization and function of TGR5 is determined by a helical structure of the membrane proximal part of the C-terminus, whereas variants

with non-helical secondary structure lead to retention in the ER and loss of function.

Reference:

[1] Johannes R. Hov, Verena Keitel, Jon K. Laerdahl, Lina Spomer, PSC research group, Schrumpf E, Häussinger D, Franke A, Karlsen TH.: Mutational Characterization of the Bile Acid Receptor TGR5 in Primary Sclerosing Cholangitis. PLoS One. 2010 Aug 25;5(8).