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Aspartate mutation distinguishes ETA but not ETB receptor subtype-selective ligand binding while abolishing phospholipase C activation in both receptors.

Authors
  • Rose, P M
  • Krystek, S R Jr
  • Patel, P S
  • Liu, E C
  • Lynch, J S
  • Lach, D A
  • Fisher, S M
  • Webb, M L
Type
Published Article
Journal
FEBS Letters
Publisher
Wiley (John Wiley & Sons)
Publication Date
Mar 20, 1995
Volume
361
Issue
2-3
Pages
243–249
Identifiers
PMID: 7698331
Source
Medline
License
Unknown

Abstract

The endothelin receptors, ETA and ETB, are G protein-coupled receptors (GPCR) that show distinctively different binding profiles for the endothelin peptides and other ligands. We recently reported that Tyr129 in the second transmembrane region (TM2) of the ETA receptor was critical for subtype-specific ligand binding [Krystek, S.R. et al. (1994) J. Biol. Chem. 269, 12383-12386]. Receptor models indicated that aspartic acids located one helical turn above (Asp133) and below (Asp126) Tyr129 in ETA had their side chains directed toward the putative binding cavity. Similarly in ETB, Asp147 and Asp154 are located one turn below and above His150, the residue that corresponds to Tyr129. Asp126 in ETA and Asp147 in ETB correspond to the highly conserved aspartate present in TM2 of many GPCR that has frequently been shown to be crucial for agonist efficacy. Mutagenesis of Asp126 of the human ETA receptor to alanine resulted in an unaltered affinity for ET-1, a 160-fold increase in ET-3 affinity and a decrease in affinity for the ETA selective naphthalenesulfonamide, BMS-182874. ET-1 activation of phospholipase C was abolished. In addition, despite the gain in binding affinity, ET-3 failed to activate phospholipase C, suggesting that Asp126 is required for signal transduction. Mutagenesis of Asp133 to alanine indicated that it was critical only for the binding of BMS-182874. In the ETB receptor, mutation of His150 to alanine or tyrosine indicated that it plays a minor role in ETB subtype-selective ligand binding; mutation of the aspartates in TM2 of ETB did not alter ligand binding. As in the Asp126 Ala ETA variant, ET-1 and ET-3 failed to increase intracellular levels of inositol phosphates in the Asp147Ala ETB mutant. Taken together, these data support the hypothesis that Asp126 and Asp133 flanking Tyr129 in TM2 of the ETA receptor play a role in defining ETA subtype-selective ligand binding but Asp147 and Asp154 that flank the His150 in TM2 of the ETB receptor do not. Furthermore, these data indicate that Asp126 in ETA and Asp147 in ETB are important for transmembrane signaling via phospholipase C.

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