ASPARTATE MUTATION DISTINGUISHES ET(A) BUT NOT ET(B) RECEPTOR SUBTYPE-SELECTIVE LIGAND-BINDING WHILE ABOLISHING PHOSPHOLIPASE-C ACTIVATION IN BOTH RECEPTORS

The endothelin receptors, ET(A) and ET(B), are G protein-coupled recep tors (GPCR) that show distinctively different binding profiles for the endothelin peptides and other ligands, We recently reported that Tyr( 129) in the second transmembrane region (TM2) of the ET(A) receptor wa s 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 (Asp(133)) and below (A sp(126)) Tyr(129) in ET(A) had their side chains directed toward the p utative binding cavity, Similarly in ET(B), Asp(147) and Asp(154) are located one turn below and above His(150), the residue that correspond s to Tyr(129). Asp(126) in ET(A) and Asp(147) in ET(B) correspond to t he highly conserved aspartate present in TM2 of many GPCR that has fre quently been shown to be crucial for agonist efficacy, Mutagenesis of Asp(126) of the human ET(A) receptor to alanine resulted in an unalter ed affinity for ET-1, a 160-fold increase in ET-3 affinity and a decre ase in affinity for the ET(A) selective naphthalenesulfonamide, BMS-18 2874, ET-1 activation of phospholipase C was abolished, In addition, d espite the gain in binding affinity, ET-3 failed to activate phospholi pase C, suggesting that Asp(126) is required for signal transduction, Mutagenesis of Asp(133) to alanine indicated that it was critical only for the binding of BMS-182874. In the ET(B) receptor, mutation of His (150) to alanine or tyrosine indicated that it plays a minor role in E T(B) subtype-selective ligand binding; mutation of the aspartates in T M2 of ET(B) did not alter ligand binding, As in the Asp(126) Ala ET(A) variant, ET-1 and ET-3 failed to increase intracellular levels of ino sitol phosphates in the Asp(147) Ala ET(B) mutant, Taken together, the se data support the hypothesis that Asp(126) and Asp(133) flanking Tyr (129) in TM2 of the ET(A) receptor play a role in defining ET(A) subty pe-selective ligand binding but Asp(147) and Asp(154) that flank the H is(150) in TM2 of the ET(A) receptor do not, Furthermore, these data i ndicate that Asp(126) in ET(A) and Asp(147) in ET(B) are important for transmembrane signaling via phospholipase C.