Ligand-dependent Differences in the Internalization of Endothelin A and Endothelin B Receptor Heterodimers

Abstract

Endothelin-1 (ET-1) is a potent vasoactive peptide that acts on endothelin A (ETEndothelins (ET-1, ET-2, and ET-3) 1 are important regulators in the vascular system. They act via two receptors: the endothelin A (ET A ) and endothelin B (ET B ) receptors (1, 2). Although human ET A and ET B receptors share 59% amino acid sequence identity (exceeding 75% at the cytoplasmic face), both receptor subtypes couple to different G proteins and differ in their ligand-induced internalization and intracellular trafficking. Whereas the ET A receptor stimulates G proteins of the G q/11 and G 12/13 families, the ET B receptor activates mainly G proteins of the G i and G q/11 families (3, 4). Whether ET B receptors also stimulate proteins of the G 12/13 family is still controversial and may depend on expression levels or cell types investigated (5, 6). Upon ligand binding, both receptor subtypes are rapidly desensitized by phosphorylation through the G protein-coupled receptor kinase type 2 (7). Following internalization via caveolae and/or clathrin-coated pits, the ET A receptor is recycled back to the cell surface (8, 9). In contrast, the ET B receptor is exclusively internalized via a clathrin-dependent pathway and transported to late endosomes and lysosomes (9, 10).The ET A receptor is mainly expressed in vascular smooth muscle cells. Its activation elicits a long-lasting contraction via an increase in cytosolic Ca 2ϩ concentrations and activation of Rho proteins (11,12). The ET B receptor is predominantly expressed in endothelial cells and stimulates the release of NO and prostacyclin, thereby causing relaxation of vascular smooth muscle cells (13). In addition, ET A and ET B receptors are co-expressed in numerous cells, e.g. astrocytes, cardiomyocytes, epithelial cells of the choroid plexus and the anterior pituitary, and certain vascular smooth muscle cells (14 -16). In disease states, such as atherosclerosis and hypercholesterolemia, vascular smooth muscle cells co-express ET A and ET B receptors (17). Because atypical ligand binding was observed for cells co-expressing ET A and ET B receptors, e.g. astrocytes, epithelial cells of the anterior pituitary, or vascular smooth muscle cells, it was suggested that the two receptor subtypes form heterodimers (15,16,18). For example, in epithelial cells of the anterior pituitary, ET B receptor-selective ligands such as sarafotoxin 6c, ET-3, and IRL1620 were competitors of 125 I-ET-1 binding only in the presence of the ET A receptor-selective antagonist BQ123 (16). In astrocytes, ET A and ET B receptors cooperatively control ET-1 clearance, because only the combi-

Dimerization of G protein-coupled receptors: New avenues for somatostatin receptor signalling, control and functioning

“…Furthermore, whereas binding of adenosine A1 and purine P2Y1 increases receptor oligomerization from a dimeric basal state (Yoshioka et al, 2002), binding of chemokines to CXCR4 promotes oligomerization from a monomeric basal state . On the other hand, it has also been reported that ligand binding induces the converse effect, so that binding of NPY, TRH and cholecystokinin to their respective receptors induces the dissociation of the assembled dimers/oligomers (Cheng and Miller, 2001;Latif et al, 2001;Berglund et al, 2003) which might be related to further endocytic processes (Gregan et al, 2004). Finally, dimeric state of some receptors remains constitutive and insensitive to ligand binding, as reported for the MTR1 receptor or the murine sst2 and sst3 (Pfeiffer et al, 2001;Ayoub et al, 2002).…”

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

“…ETA and ETB and are co-expressed in a variety of cells, including ventricular myocytes [38], and can form heterodimers that may differ from receptor monomers, and possibly homodimers, with respect to endocytosis or trafficking itineraries [42,43]. As aortic endothelial cells express only ETB, rat aortic endothelial cells were used to determine if the absence of ETA altered the trafficking of ET:ETB complexes (Figure 4).…”

Pharmacochaperones Post-translationally Enhance Cell Surface Expression by Increasing Conformational Stability of Wild-type and Mutant Vasopressin V2 Receptors

“…Transient transfection was carried out with LipofectAMINE or with FuGENE 6 essentially as described (9,11). For co-transfections of plasmids encoding the mV 2 R or the hV 2 R mutants with plasmids encoding wild-type or mutant dynamin, the ratio of DNA was 0.7/0.3.…”

“…LSM-FRET imaging and fluorescence recovery after photobleaching were performed as described in detail previously (11,14). LSM-FRET imaging was performed with the LSM510 META and for fluorescence recovery after photobleaching we used an inverted microscope (Axiovert 100; Zeiss) equipped with a Plan-Apochromat 63ϫ/1.4 objective, a monochromator (Polychrome II; TILL Photonics, Grä felfing, Germany) and a cooled CCD camera (Imago; TILL Photonics).…”