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) di splaying an ET-1-selective profile and ET(B) a nonselective one. To id entify the regions responsible for the differential selectivity, chime ric forms were engineered by sequentially exchanging extracellular reg ions together with their flanking transmembrane domains. Two sets of r eciprocal receptor mutants were thereby generated and analysed by expr ession in COS-7 cells. The recombinant receptor chimeras were characte rised by direct and competitive radioligand-binding analysis. COS-7 ce lls transfected with vectors for the mutant receptors exhibited specif ic saturable [3-I-125]iodotyrosyl ET-1 (I-125-ET-1) binding, with affi nities comparable to those of the wild-type receptors (apparent K-i ap proximate to 1-6 x 10(-9)M). An average of 10(5)-10(6) binding sites/c ell 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 ag onist, we detected a clear switch from an ET-1-selective profile to a non-isopeptide-selective profile in ETS chimeras where the second extr acellular 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 opp osite 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), w e were able to map the main determinants responsible for this selectiv ity to the N-terminal moiety of this receptor. Therefore, the ability for the interaction with ET-3 or BQ123 is governed by two different re gions of the ET receptors.