Palmitoylation of Endothelin Receptor A

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...

Section: Internalization and Intracellular Trafficking Of Et Receptormentioning

confidence: 80%

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

Paasche

Attramadal

Sandberg

et al. 2001

“…In many cases, these have been shown directly to be sites for palmitoylation (36,(52)(53)(54)(55). Mutation of these residues has been reported to have effects ranging from alterations in G protein coupling (52)(53)(54) and receptor internalization/ membrane delivery (55,56) to effects on the phosphorylation of the GPCR by regulatory kinases (36,57) and interactions with ␤-arrestin-1 (58). The ␣ 1b -adrenoreceptor-C9S,C10S G␣ 11 fusion protein was also able to incorporate [ 3 H]palmitate.…”

Section: Discussionmentioning

confidence: 99%

Coordinated Agonist Regulation of Receptor and G Protein Palmitoylation and Functional Rescue of Palmitoylation-deficient Mutants of the G Protein G11α following Fusion to the α1b-Adrenoreceptor

Stevens¹,

Pediani

Carrillo

et al. 2001

Transfection of either the ␣ 1b -adrenoreceptor or G␣ 11 into a fibroblast cell line derived from a G␣ q /G␣ 11 double knockout mouse failed to produce elevation of intracellular [Ca 2؉ ] upon the addition of agonist. Co-expression of these two polypeptides, however, produced a significant stimulation. Co-transfection of the ␣ 1b -adrenoreceptor with the palmitoylation-resistant C9S,C10S G␣ 11 also failed to produce a signal, and much reduced and kinetically delayed signals were obtained using either C9S G␣ 11 or C10S G␣ 11 . Expression of a fusion protein between the ␣ 1b -adrenoreceptor and G␣ 11 allowed [Ca 2؉ ] i elevation, and this was also true for a fusion protein between the ␣ 1b -adrenoreceptor and C9S,C10S G␣ 11 , since this strategy ensures proximity of the two polypeptides at the cell membrane. For both fusion proteins, co-expression of transducin ␣, as a ␤⅐␥-sequestering agent, fully attenuated the Ca 2؉ signal. Both of these fusion proteins and one in which an acylation-resistant form of the receptor was linked to wild type G␣ 11 were also targets for agonist-regulated

“…Protein lipidation appears at numerous steps in the process of signaling through heterotrimeric G proteins. Many GPCRs are subject to palmitoylation within their carboxyl-terminal cytoplasmic C-tail region (63)(64)(65)(66); it has been proposed that the presence of the carbon 16 saturated fatty acyl group may anchor a portion of the C-tail to the plasma membrane, thereby forming a fourth intracellular loop in this region (29). In at least one case, that of the ␤ 2 adrenergic receptor, the palmitoylation state of the receptor is influenced by agonist occupation (67).…”

Section: Discussionmentioning

confidence: 99%

The Prostacyclin Receptor Is Isoprenylated

et al. 1999

The prostacyclin receptor (IP), a G protein-coupled receptor, mediates the actions of the prostanoid prostacyclin and its mimetics. IPs from a number of species each contain identically conserved putative isoprenylation CAAX motifs, each with the sequence CSLC. Prostacyclin (prostaglandin (PG)1 I 2 ) is a labile metabolite of arachidonic acid, which is synthesized by the sequential actions of PGH 2 endoperoxide synthases 1 and 2 and prostacyclin synthase (1). The actions of prostacyclin generally counteract those of thromboxane A 2 , and thus the relative level of these two prostanoids in the circulation are important in the local control of vascular tone and platelet aggregation (2, 3). The main physiologic roles of thromboxane A 2 and prostacyclin are their contribution to the maintenance of vascular hemostasis: thromboxane A 2 , synthesized primarily by platelets, induces platelet shape change and aggregation and constriction of bronchial and vascular smooth muscle, whereas prostacyclin, mainly synthesized by the vascular endothelium, is a potent inhibitor of platelet aggregation and induces vasodilation (4 -7). Moreover, prostacyclin has been reported to confer a cytoprotective effect against tissue injury in acute myocardial ischemia or following hypoxic exposure of vascular endothelial cells (8). Imbalances in thromboxane A 2 or prostacyclin have been reported to be a major contributing factor in the development of a number of cardiovascular disorders including thrombosis, myocardial infarction, unstable angina, stroke, and atherosclerosis (9 -13). In addition to its central role in the cardiovascular system, prostacyclin may be important in the regulation of renal blood flow (14); it also acts as a negative feedback regulator of histamine secretion from mast cells (15) and acts as a lipolytic agent, antagonizing the antilipolytic effect of PGE 2 , in adipocytes (16).The actions of prostacyclin are mediated via interaction with a specific cell surface receptor, termed the prostacyclin receptor or IP (17). The major intracellular signaling pathway used is stimulation of adenylyl cyclase leading to increases in intracellular cAMP (18,19), a pathway thought to be relevant to inhibition of platelet aggregation and vascular smooth muscle relaxation (3). However, recent evidence indicates that IP agonists may couple to multiple signaling pathways including activation and inhibition of adenylyl cyclase, via G s and G i , respectively, stimulation of phosphoinositide metabolism, and changes in [Ca 2ϩ ] i concentrations (20,21). Evidence also exists to indicate that iloprost, a stable carbacyclin analogue of prostacyclin, can stimulate opening of ATP sensitive K ϩ channels resulting in hyperpolarization and relaxation of canine carotid artery (22).Molecular cloning of the human (23, 24), mouse (25), and rat