Subtype-Specific Sorting of the ET_AEndothelin Receptor by a Novel Endocytic Recycling Signal for G Protein-Coupled Receptors

Abstract

We have previously reported that endocytic sorting of ET A endothelin receptors to the recycling pathway is dependent on a signal residing in the cytoplasmic carboxyl-terminal region. The aim of the present work was to characterize the carboxylterminal recycling motif of the ET A receptor. Assay of truncation mutants of the ET A receptor with increasing deletions of the carboxyl-terminal tail revealed that amino acids 390 to 406 contained information critical for the ability of the receptor to recycle. This peptide sequence displayed significant sequence similarity to several protein segments confirmed by X-ray crystallography to adopt antiparallel ␤-strand structures (␤-finger). One of these segments was the ␤-finger motif of neuronal nitric-oxide synthase reported to function as an internal PDZ (postsynaptic density-95/disc-large/zona occludens) domainbinding ligand. Based on these findings, the three-dimensional structure of the recycling motif of ET A receptor was predicted to attain a ␤-finger conformation acting as an internal PDZ ligand. Site-directed mutagenesis at residues that would be crucial to the structural integrity of the putative ␤-finger conformation or PDZ ligand function prevented recycling of the ET A receptor. Analysis of more than 300 G protein-coupled receptors (GPCRs) identified 35 different human GPCRs with carboxylterminal sequence patterns that fulfilled the structural criteria of an internal PDZ ligand. Among these are several receptors reported to follow a recycling pathway. In conclusion, recycling of ET A receptor is mediated by a motif with the structural characteristics of an internal PDZ ligand. This structural motif may represent a more general principle of endocytic sorting of GPCRs.The physiological effects of the vasoactive peptide endothelin-1 (ET-1) are mediated by the ET A and ET B receptors, which belong to class A G protein-coupled receptors (GPCRs) (Yanagisawa et al., 1988). In the vasculature, ET A receptors residing on smooth muscle cells mediate prolonged vasoconstriction, whereas ET B receptors, which are located on the plasma membrane of endothelial cells, are primarily considered to cause NO-mediated vasodilation (Yanagisawa and Masaki, 1989). In addition, considerable evidence now also supports a role for the ET B receptor in clearance of plasma ET-1 from the circulation (Berthiaume et al., 2000;Opgenorth et al., 2000). We have previously shown that agonist-induced internalization of the two receptor subtypes depends on a mechanism involving G protein-coupled receptor kinase, arrestin, clathrin, and dynamin (Bremnes et al., 2000). After internalization, however, the two receptor subtypes are targeted to different intracellular fates. The ET A receptor follows the recycling pathway through the pericentriolar recycling compartment and subsequently reappears on the plasma membrane, whereas the ET B receptor is directed to lysosomes for degradation (Bremnes et al., 2000). In terms of physiological effects, rapid recycling of the ET A receptor may provide ba...

An intact PDZ motif is essential for correct P2Y12 purinoceptor traffic in human platelets

“…28 In addition to C-terminal PDZ ligands, one study has indicated that an internal PDZ ligand present in the ET A endothelin receptor C-tail can regulate receptor recycling. 29 Our study has also shown that the C-terminal ETPM motif of the P2Y 12 R, a type I PDZ motif, is required for receptor recycling. Deletion or mutation of this sequence leads to the accumulation of the receptor in an intracellular sorting compartment and blockade of receptor recycling.…”

G Protein–Coupled Receptor Sorting to Endosomes and Lysosomes

“…A subset of VHS domains have been shown to bind to acidic diLeu motifs; however a direct interaction between the VHS domain of HRS and the acidic diLeu motif of the β 2 AR is unlikely because the VHS domain lacks the critical residues important for binding to acidic di-Leu D-X-X-L-L motifs (46,47). In addition to C-terminal PDZ ligands, one study has indicated that an internal PDZ ligand present in the ET A endothelin receptor Ctail can regulate receptor recycling (48), but the endocytic sorting machinery that mediates this process has not been identified. Interestingly, internal PDZ ligand sequences have also been identified in several other human GPCRs (48).…”

“…In addition to C-terminal PDZ ligands, one study has indicated that an internal PDZ ligand present in the ET A endothelin receptor Ctail can regulate receptor recycling (48), but the endocytic sorting machinery that mediates this process has not been identified. Interestingly, internal PDZ ligand sequences have also been identified in several other human GPCRs (48).…”

Sarcoplasmic-endoplasmic reticulum Ca2+-ATPase inhibition prevents endothelin A receptor antagonism in rat aorta

“…Caveolar structures cluster signaling molecules through caveolin and PDZ (postsynaptic density protein, Drosophila disc large tumor supressor, Zo-1 protein) domain proteins in a spatially confined subsarcolemmal region to make protein-protein interactions more rapid and efficient (34). A recent study (20) also suggests that internal PDZ ligand motif seems to regulate efficient recycling of the ET A endothelin receptor. Later, growing evidence suggested that caveolin-1 regulates the interaction of ET-1 with its receptors (37).…”