The hydrophobic amino acid residues in the membrane‐proximal C tail of the G protein‐coupled vasopressin V2 receptor are necessary for transport‐competent receptor folding

Abstract

It is believed that the membrane-proximal C tail of the G protein-coupled receptors forms an additional alpha helix with amphipathic properties (helix 8). It was previously shown for the vasopressin V2 receptor (V2R) that a conserved dileucine motif (L 339 , L 340 ) in this putative helix 8 is necessary for endoplasmic reticulum (ER) to Golgi transfer of the receptor. Here, we demonstrate that the other hydrophobic residues forming the non-polar side of this helix (F 328 , V 332 and L 336 ) are also transport-relevant. In contrast, the multiple serine residues contributing to the more hydrophilic side (S 330 , S 331 , S 333 , S 334 , S 338 ) do not influence receptor trafficking. In addition, we show unambiguously by the use of pharmacological chaperones that the hydrophobic residues of the putative helix 8 do not form a transport signal necessary for receptor sorting into ER to Golgi vesicles. Instead, they are necessary to establish a transportcompetent folding state in the early secretory pathway.

A Conserved Motif in the Membrane Proximal C-Terminal Tail of Human Muscarinic M1Acetylcholine Receptors Affects Plasma Membrane Expression

“…The C-terminal leucines of the F(x) 6 LL motif (L 339 and L 340 ) of V2 receptors may interact with residues at the base of TM1 (Thielen et al, 2005). We were interested in determining, therefore, whether hM 1 receptors containing mutations at the base of TM1 (hM ) associate with the ER in a manner similar to that of hM .…”

“…In the vasopressin V2 receptor, the F(x) 6 LL motif exists in an amphipathic ␣-helix referred to as helix 8 (Thielen et al, 2005). The C-terminal leucines of the F(x) 6 LL motif (L 339 and L 340 ) of V2 receptors may interact with residues at the base of TM1 (Thielen et al, 2005).…”

“…, or L 340 in this motif caused ER retention of the receptor (Krause et al, 2000;Thielen et al, 2005). The membrane-permeable V 2 receptor antagonist SR121463B rescued the plasma membrane expression (Thielen et al, 2005), suggesting that the mutant V 2 receptor could not achieve a transport-competent conformation (Krause et al, 2000;Thielen et al, 2005). Perhaps the F(x) 6 LL motif is another type of folding motif that mediates transport-competent folding necessary for many GPCRs to exit the ER and enter the secretory pathway.…”

The physiological and pathophysiological functions of renal and extrarenal vasopressin V2 receptors

“…In addition, V2R is palmitoylated at cysteines C341 and C342, stabilizing the protein in the membrane (101). When the receptor is correctly folded, it will transit via the intracellular membrane systems to the cell surface (117).…”

“…1, three intracellular loops and three extracellular loops connect seven transmembrane helixes. There is an extracellular NH 2 terminus, which is involved in ligand binding, and the signaling activity of the GPCR is enabled by the intracellular COOH terminus and the third intracellular loop (100,101,104,117). The receptor has a complex three-dimensional structure that is generated in the endoplasmic reticulum (ER) and Golgi apparatus by posttranslational sugar moieties at N22, COOH-terminal serines, and/or threonines (100) as well as two conserved cysteine residues (putative disulfide bonds) between C112 in the first extracellular loop and C192 in the second extracellular loop (104).…”