The proximal TGR5 C-Terminus is required for membrane localization

TGR5 (Gpbar–1) is a plasma membrane-bound bile acid receptor, which is coupled to a stimulatory G-protein. The structure of TGR5 is unknown and it remains elusive which amino acids are important for receptor localization and function. Based on previous findings [1] that a C-terminal truncation variant (Q296) is retained in the endoplasmic reticulum and therefore nonfunctional, we tried to identify motifs in the TGR5 C-terminus important for receptor localization and function.

Due to our previous findings we generated further truncation variants (D284, P290, A295, R297, Q300 and S310). The truncation variants D284, P290, A295 and R297 were localized in the endoplasmic reticulum (ER). The variants Q300 and S310 were detected in both the ER as well as in the plasma membrane (PM). Accordingly the variants D284, P290, A295 and R297 completely lost responsiveness to TLC, while TGR5 receptor activity was reduced or unaffected by the absence of the last 30 or 20 amino acids, respectively. To analyze the importance of amino acid 284 to 297 for receptor localization, TGR5 deletion variants (Δ284–290, Δ291–297) and TGR5 alanine substitution variants (285–290A and 291–297A) were generated. The deletion variants and 285–290A were mainly localized in the ER and unresponsive to TLC stimulation. The 291–297A variant was detected both in the ER as well as in the PM. TGR5 function of this variant was slightly reduced.

Truncation of TGR5 at amino acids 284–297 leads to the accumulation of the mutated proteins in the ER and reduces responsiveness to TLC. The deletion variants and alanine substitutions indicate that amino acids 284–290 are important for receptor localization and consequently for function. The proximal C-terminal region could act as a signalling motif for the export of the protein from the ER and therefore determine cell-surface expression and function. Another explanation is that the deletion of amino acids 284–290 impairs protein folding thus leading to ER retention.

Literatur: 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).