To understand the biochemical basis for the functional divergence of the human endothelin receptor subtypes A (ETAR) and B (ETBR), they were expressed, purified from insect Sf9 cells, and reconstituted into phospholipid vesicles with the Go, Gq, and Gi proteins. For each G protein, a unique pattern of reactivity was observed with the different receptor subtypes. Both ETAR and ETBR activated Go to a similar maximal extent, and both subtypes activated Gq with similar EC50 values; however, the ETAR displayed a 2-3-fold higher maximal extent of activation. In contrast, both subtypes activated Gi to a similar maximal extent, but the ETAR displayed a 4-fold higher EC50 value as compared to the ETBR. To test whether these coupling specificities are influenced by C-terminal palmitoylation of the receptor, we mutated a cluster of cysteine residues near the end of the seventh transmembrane helix in both receptors. While the cysteine mutations in the ETBR resulted in a partially palmitoylated receptor, the replacement of these cysteine residues in the ETAR yielded a mostly palmitoyl-deficient receptor and had no effect on Go activation, but caused a reduction in the extents of Gi and Gq stimulation. Together, these studies provide important insights into the specificity of G protein coupling in the endothelin receptors. The ability to discriminate between the different G proteins under various physiological conditions may be a key element in the selection of distinct signal transduction pathways by the two receptor subtypes.
Endothelin type-B receptor (ET,R) forms a stable complex with its ligand, endothelin-I. To facilitate biochemical and biophysical studies of human ET,R, several ET,R mutants carrying a hexahistidine tag sequence at the N or C terminus were expressed in Sf9 cells and were purified by a combination of biotinylated endothelin-1 -1igand-affinity and nickel-affinity chromatographies. The ligand-free receptor was purified by dissociating the ligand . receptor complex with 2 M NaSCN, whereas the ligand-bound ET,R was purified by the use of thiol-sensitive biotinylated endothelin-I. While the wild-type ET,R was expressed at about 100 pmol '251-endothelin-l -binding activity/mg membrane protein, the deletion of 36 residues from the N-terminus reduced the expressed activity to about 30%. On the other hand, the lack of glycosylation and the replacement of 2-9 residues in the N-terminal tail resulted in a 20-40% reduction in the expressed activity. Among the mutant proteins, [H57-H62, G63 -G65]ETBR, carrying six His residues in the N-terminal tail, was studied extensively because it was purified most effectively. Ligand-free [H57 -H62, G63 -G65]ETBR, purified in digitonin, retained full ligand-binding activity, while other detergents led to partial denaturation of the receptor after solubilization or after elution with NaSCN. On the other hand, ligand-bound [H57-H62, G63 -G65]ETBR could be purified in various detergents, such as n-octyl-P-D-glucopyranoside or n-decyl-/b-maltopyranoside. Ligand-free [H57 -H62, G63 -G65]ETBR reconstituted in phospholipid vesicles stimulated the binding of guanosine 5'-3-0-(thi0)triphosphate by G, in the presence of endothelin-I. Ligand-bound [H57 -H62, G63 -G65]ETBR showed similar catalytic activity in nucleotide exchange by G,. These results indicate that the ligand . receptor complex in a detergent-micellar solution retained the biologically active structure, and that the presence of ligand, endothelin-1, in the receptor molecule reinforces the stable assembly of a helical bundle and therefore the active structure.Keywords: endothelin ; endothelin B receptor; guanine-nucleotide-binding regulatory protein ; reconstitution.Guanine-nucleotide-binding regulatory protein (G protein)-coupled receptors are cell-surface membrane proteins that mediate various signals through G proteins [l, 21. They are considered to have a common structural motif of a membrane-spanning heptahelical bundle [3], and cytoplasmic and extracellular domains. Many studies have shown that the interaction sites for ligands are in the transmembrane domain, and that G proteins bind to the cytoplasmic domain, In particular, the binding pockets for small ligands, such as the catecholamines for the adrenergic receptor and acetylcholine for the muscarinic acetylcholine receptor, are thought to be in the transmembrane helical cluster [4], similar to the binding of retinal in rhodopsin [3, 5, 61 and bacteriorhodopsin [7]. However, the binding pockets for many ligands, such as peptide and glycoprotein hormones, appear to Correspond...
The mutation of W276 to cysteine within the human endothelin receptor subtype B (ET(B)R) is associated with Hirschsprung's disease, a congenital intestinal disease. The sequence surrounding W276 is highly conserved between the endothelin receptor subtypes A and B. We have introduced sets of mutations into W275 and W276 of the ET(B)R gene, and the corresponding W257 and W258 of the ET(A)R gene, and studied their coupling properties with G(i), G(o), and G(q) in reconstituted phospholipid vesicles. The prepared mutants all showed a similar affinity for endothelin-1. The W276C/ET(B)R and W276A/ET(B)R mutants had reduced activities in G(q) coupling but not in G(i)/G(o) coupling, while the W275A/ET(B)R displayed reduced activities in G(i)/G(q) coupling, with normal G(o) coupling. On the other hand, W257A/ET(A)R and W258A/ET(A)R exhibited wild-type activities in all examined G protein couplings. These results suggest that the defects in the G(q) signaling pathway by the ET(B)R are connected with Hirschsprung's disease and that the two conserved tryptophans play distinct roles in signal transduction by the two receptor subtypes. In addition, W275 and W276, which are thought to be located near the extracellular side of the transmembrane helix 5, play important roles in forming the active structure of ET(B)R.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.