MUTATIONAL ANALYSIS OF HUMAN ENDOTHELIN RECEPTORS ET(A) AND ET(B) - IDENTIFICATION OF REGIONS INVOLVED IN THE SELECTIVITY FOR ENDOTHELIN-3 OR CYCLO-(D-TRP-D-ASP-PRO-D-VAL-LEU)

Two endothelin(ET)-receptor subtypes have been identified in mammals. They differ in their affinity towards the ET isopeptides with ET(A) displaying an ET-1-selective profile and ET(B) a nonselective one. To identify the regions responsible for the differential selectivity, chimeric forms were engineered by sequentially exchanging extracellular regions together with their flanking transmembrane domains. Two sets of reciprocal receptor mutants were thereby generated and analysed by expression in COS-7 cells. The recombinant receptor chimeras were characterised by direct and competitive radioligand-binding analysis. COS-7 cells transfected with vectors for the mutant receptors exhibited specific saturable [3-I-125]iodotyrosyl ET-1 (I-125-ET-1) binding, with affinities comparable to those of the wild-type receptors (apparent K-i approximate to 1-6 x 10(-9)M). An average of 10(5)-10(6) binding sites/cell was calculated for the wild-type and mutant forms. In competition experiments using I-125-ET-1 and unlabeled ET-3, an ET(B)-selective agonist, we detected a clear switch from an ET-1-selective profile to a non-isopeptide-selective profile in ETS chimeras where the second extracellular loop and the flanking transmembrane domains IV and V or the third extracellular loop and the flanking transmembrane domains VI and VII, had been exchanged for the corresponding parts of ET(B). The opposite effect, namely a switch from a non-isopeptide-selective to an ET-1-selective binding, was observed for the mirror ET(A) chimeras where the symmetrical exchange had been operated. Using I-125-ET-1 and the ET(A)-specific antagonist cyclo-(D-Trp-D-Asp-Pro-D-Val-Leu) (BQ123), we were able to map the main determinants responsible for this selectivity to the N-terminal moiety of this receptor. Therefore, the ability for the interaction with ET-3 or BQ123 is governed by two different regions of the ET receptors.