A Dileucine Sequence and an Upstream Glutamate Residue in the Intracellular Carboxyl Terminus of the Vasopressin V₂Receptor Are Essential for Cell Surface Transport in COS.M6 Cells

Abstract

Little is known concerning the intracellular transport of the G protein-coupled receptors (GPCRs). Previous studies suggested a functional role for those residues immediately preceding the conserved palmitoylated cysteine residues in the intracellular carboxyl termini of some GPCRs in cell surface transport. For the human vasopressin V2 receptor, we assessed the significance of a dileucine sequence with an upstream glutamate residue (ELRSLLCC) in mediating cell surface delivery. A series of deletion and point mutants in this region were constructed, and the mutant receptors were expressed in transiently transfected COS.M6 cells. By using [3H]arginine vasopressin binding assays to intact cells and immunofluorescence studies with intact and permeabilized cells, we show that residues E335 (mutant E335Q) and L339 (mutant L339T) are obligatory for receptor transport to the plasma membrane. Residue L340 has a minor but significant influence. [3H]Arginine vasopressin binding experiments on membranes of lysed cells failed to detect any intracellular binding sites for the transport-deficient mutant receptors, suggesting that residues E335 and L339 participate in receptor folding. Studies with green fluorescent protein-tagged receptors demonstrate that the bulk of the mutant receptors E335Q and L339T are trapped in the endoplasmic reticulum. Complex glycosylation was absent in these mutant receptors, supporting this conclusion. These data demonstrate that the glutamate/dileucine motif of the vasopressin V2 receptor is critical for the escape of the receptor from the endoplasmic reticulum, most presumably by establishing a functional and transport-competent folding state. A databank analysis revealed that these residues are part of a conserved region in the GPCR family.

Cell Surface Expression of the Melanocortin-4 Receptor Is Dependent on a C-terminal Di-isoleucine Sequence at Codons 316/317

“…Previous work (18) has indicated that the di-leucine motif in the C terminus of the vasopressin V 2 receptor is important in the exiting of the receptor from the endoplasmic reticulum. In that study, single substitutions of hydrophobic leucines by hydrophobic isoleucine had no effect on V 2 receptor cell surface expression (18); however, substitution of either leucine by the polar threonine decreased cell surface expression, and the double substitution completely suppressed it.…”

“…Acidic dihydrophobic motifs, in particular di-leucine pairs preceded by a cluster of acidic amino acids, have been shown to be sorting receptor binding sites for the GGA (Golgi-localizing, ␥-adaptin ear homology domain, ARF-interacting) proteins required for the transport of protein cargo from the trans-Golgi network to endosomes (24,25). The C-terminal dihydrophobic motif that appears in GPCRs is preceded, in many GPCRs, by a conserved acidic amino acid (18). Moreover, substitution of this residue (glutamate) by glutamine in the vasopressin V 2 receptor also blocks cell surface expression of the receptor (18).…”

“…This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. cine, phenylalanine, valine, or methionine and preceded by an acidic glutamate or aspartate (18). Mutation of the conserved dileucine motifs in the vasopressin V 2 and lutropin/choriogonadotropin receptors have been shown to limit membrane targeting of these receptors (18 -20).…”

SOCS-mediated downregulation of mutant Jak2 (V617F, T875N and K539L) counteracts cytokine-independent signaling

“…Confocal live-cell imaging (at 37 1C in 5% CO 2 in a phenol red-free medium) was carried out using a Zeiss LSM510 invert laser scanning microscope (Zeiss, Sliedrecht, Netherlands). YFP was detected with l excitation ¼ 488 nm and l emission ¼ 515À550 nm, cell surface was stained with 0.05% trypan blue (Schulein et al, 1998) and recorded with l excitation ¼ 543 nm and l emission >590 nm.…”

Regulation of anterograde transport of adrenergic and angiotensin II receptors by Rab2 and Rab6 GTPases

“…It has been demonstrated that export from the ER is a rate-limiting step for maturation of the δ-opioid receptor [8], and accumulation of misfolded receptors in the ER is associated with the pathogenesis of human disease [9,10]. Second, GPCR export from the ER is directed by highly conserved motifs in the membrane-proximal termini [11][12][13][14][15]. We have recently identified the F(X) 6 LL motif in the C-termini required for α 2B -adrenergic (α 2B -AR) and angiotensin II type 1 receptor (AT1R) export from the ER [14,15] and the YS motif in the N-termini essential for α 2 -AR export from the Golgi [16].…”