Mechanisms of Endothelin Receptor Subtype-specific Targeting to Distinct Intracellular Trafficking Pathways

Abstract

We recently reported that the endothelin (ET) receptor subtypes ET 275, 17596 -17604). The ET A receptor was shown to follow the recycling route of transferrin, whereas ET B is targeted to lysosomes for degradation. In the present study we have investigated the mechanisms of ET receptor subtype-specific targeting to distinct intracellular trafficking pathways. Truncation mutants of the ET A and ET B receptors with deletions of the cytoplasmic carboxyl-terminal tail distal to the palmitoylation site were found to mediate inositol phosphate accumulation and to internalize upon agonist stimulation, although internalization occurred at a slower rate as compared with the wild-type receptors. However, the truncated ET A receptor was no longer able to undergo recycling. Rather, both truncation mutants were recognized by ␤-arrestin for recruitment to endocytosis and were sorted to lysosomes by a dynamin-dependent internalization pathway. Furthermore, studies of chimeric ET A and ET B receptors where the cytoplasmic tail of ET A was swapped with the corresponding domain of ET B , and vice versa, revealed that the cytoplasmic tail of ET B is required for efficient lysosomal sorting and that signals for targeting to recycling reside in the cytoplasmic tail of the ET A receptor.The multiple physiological effects of the vasoactive peptide endothelin (ET) 1 (1) are mediated by the G protein-coupled receptors (GPCRs) ET A and ET B (2). In the vasculature, ET A receptors residing on the smooth muscle cells mediate prolonged vasoconstriction (3), whereas ET B receptors, which are on the plasma membrane of endothelial cells, are primarily considered to cause NO-mediated vasodilatation (4). In addition, considerable evidence now also supports a role for ET B receptors in the clearance of plasma ET-1 from the circulation (5-8). In order to elucidate the molecular mechanisms of these distinct physiological responses, we recently characterized the intracellular trafficking pathways of the ET A and ET B receptors (9). Upon agonist stimulation both receptor subtypes are rapidly internalized by mechanisms that depend on G proteincoupled receptor kinase, arrestin, clathrin, and dynamin. Interestingly, the internalized ET A and ET B receptors initially appear to share a common path into Rab5-positive early endosomes. However, the two receptor subtypes are subsequently targeted to different intracellular fates. Whereas the ET A receptor follows the recycling pathway through the pericentriolar recycling compartment and reappears at the plasma membrane, the ET B receptor is directed to lysosomes for degradation. In terms of physiological effects, rapid recycling of the ET A receptor may provide the basis for reestablishment of the signaling response, and thus for the sustained vasoconstriction mediated through this receptor. Conversely, lysosomal targeting of the ET B receptor is consistent with a role for this receptor subtype in the clearance of ET-1 from the circulation. In this respect, it was recently also demonstrated that ET-1 is cotr...

Intracrine endothelin signaling evokes IP3-dependent increases in nucleoplasmic Ca2+ in adult cardiac myocytes

“…The fate of each receptor subtype is under the control of specific elements regulating protein trafficking. Chimeric ETA and ETB constructs reveal the cytoplasmic C-terminal domain of ETA is sufficient to specify receptor recycling whereas the comparable domain in ETB specifies delivery to lysosomes [4,6]. In ventricular myocytes [12] and mouse spiral ganglion neurons [57], which express both ETA and ETB, ETB is primarily associated with the nuclear membranes whereas ETA is at the cell surface.…”

“…In heterologous expression systems, ETA and ETB differ in their internalization and intracellular trafficking. ETA shows ligand-dependent endocytosis and is recycled back to the plasma membrane whereas ETB internalizes constitutively and translocate to lysosomes [4][5][6]. Thus, it has been proposed that ETA, the prevalent receptor subtype in the cardiac tissue, mediates the main cellular functions of endothelin, whereas ETB is involved in the clearance of ET-1.…”

Agonist-promoted Ubiquitination Differentially Regulates Receptor Trafficking of Endothelin Type A and Type B Receptors

“…With respect to ETRs, this conclusion is based on the studies using chimeric mutants of ETRs where the C-tails of ET A R and ET B R were swapped (15,21). The chimeric ET B R with the C-tail of ET A R was capable of recycling like wild type ET A R, whereas the chimeric ET A R with the C-tail of ET B R behaved like wild type ET B R (15,21). However, the detailed mechanism for the different fates of ET A R and ET B R is at present totally unknown.…”

“…␤-Arrestins have been shown to function as adaptors for ubiquitination of GPCRs, such as ␤ 2 -adrenergic receptor (␤ 2 AR) (27,28), whereas both ETRs are known to recruit ␤-arrestins (18,21). Therefore, we examined a role of ␤-arrestins in ubiquitination of ET B R. Knockdown of ␤-arrestin 1 and ␤-arrestin 2 was without effect on basal and ET-1-induced ubiquitination of ET B R, demonstrating that ␤-arrestins play no significant role in ubiquitination of ET B R (Fig.…”

“…For that purpose, we overexpressed a FLAG-tagged dominant negative mutant of rat dynamin (DNdynamin) to inhibit ET B R internalization (21) and examined its effect on ET B R ubiquitination. In control cells transfected with empty vector alone, cell surface HA-ET B R rapidly decreased following ET-1 stimulation (Fig.…”