Helix 8 of leukotriene B4type‐2 receptor is required for the folding to pass the quality control in the endoplasmic reticulum

Abstract

Many G protein-coupled receptors (GPCRs) possess a putative cytoplasmic helical domain, termed helix 8 (H8), at the proximal region of the C-terminal tail. However, the significance of this domain is not fully understood. Here, we demonstrate the requirement of H8 for the proper folding of GPCRs for passage through the quality control in the endoplasmic reticulum (ER). In the human leukotriene B(4) type-2 receptor (hBLT2), lack of H8 led to an accumulation of the receptor (hBLT2/DeltaH8) in the ER. Similar results were obtained in two representative human GPCRs, dopamine type-1 and lysophosphatidic acid type-2 receptors, which were engineered to lack H8. Treatment with the several ligands, which act as pharmacological chaperones, facilitated the surface expression of hBLT2/DeltaH8. The surface-trafficked hBLT2/DeltaH8 exhibited an agonist-evoked increase in Ca(2+), demonstrating that H8 is not critical for ligand binding and activation of coupled G proteins. Thus, these results suggest that the H8 region of hBLT2 plays an important role in transport-competent receptor folding.

Specific ligands as pharmacological chaperones: The transport of misfolded G‐protein coupled receptors to the cell surface

“…Because these critical residues partially or totally constitute the putative H8 domain of each receptor, these data support our hypothesis that the H8, especially the hydrophobic residues, is important for GPCRs to pass the ER quality control system. A molecular model of BLT2 readily supports our hypothesis (21). Our model predicts that Phe 303 , Leu 304 , Leu 307 , and Phe 308 form the hydrophobic region of H8 that interacts with some residues in the TM1 and TM7 of BLT2.…”

“…Human BLT2 that lacks the H8 (BLT2/DH8) accumulates in the ER, suggesting that the H8 domain is necessary for its cell surface expression ( Fig. 3A) (21). Similarly, other human GPCRs such as the dopamine D1 and LPA type-2 receptors (LPA2) also require the H8 for their export from the ER.…”

“…Consistently, the crystal structure of rhodopsin suggests that the Asp in TM2 makes a hydrogen bond with Asn in TM1 and Ala in TM7 (7); thus, it too might contribute to the correct folding of GPCRs. (21). (B) C-terminal sequences of human (h)BLT2, rat (r)D1R, hV2R, hV1b/3R, rAT1R, and ra 2B -AR.…”

“…(C) After transfection of BLT2/WT or BLT2/DH8 into HeLa cells, the cells were treated without or with the indicated ligands for 24 h. Then, the cell surface expression levels of each receptor were determined by flow cytometric analysis. For more detail, see (21).…”

Role of Helix 8 of the Thyrotropin-Releasing Hormone Receptor in Phosphorylation by G Protein-Coupled Receptor Kinase

“…Although basic clusters in the helix 8 region are required for folding and trafficking of several receptors (Huynh et al, 2009), substitution of charged residues in this segment of the TRH and ␤2-adrenergic receptors does not appreciably alter surface expression. Deletion of hydrophobic residues in the helix 8 region of the leukotriene B 4 type-2 receptor also results in an intracellularly trapped receptor, but no trafficking defect is observed when only the polar residues are mutated (Yasuda et al, 2009). In a few cases, alterations in helix 8 cause constitutive activity and in one, constitutive phosphorylation (Faussner et al, 2005;Suvorova et al, 2009).…”