ALANINE SCAN OF ENDOTHELIN - IMPORTANCE OF AROMATIC RESIDUES

A systematic approach to map the functional important determinants of endothelin-1 (ET) by an alanine scan is described. Studies on the in v itro receptor binding affinity and on the agonist contracting activity defined that residues Asp(8), Tyr(13), Phe(14), Leu(17), and Trp(21) were of major biological significance. A striking observation was that four out of these five sites were hydrophobic amino acids. Ala analog ues of the aromatic residues at position 13, 14 and 21 displayed sharp ly reduced receptor binding affinity (<2% of ET) and can be considered important for receptor contact. Ala analogues of Asp(8) and Leu(17) l ost most (<90%) of the agonist activity but retained a receptor affini ty nearly equivalent to ET and can be considered to be important for s ignal transduction. Three other positions, Val(12), Asp(18), and Ile(2 0) (which are adjacent to the biologically important sites of Tyr(13), Leu(17), and Trp(21)), resulted as partially tolerant to Ala substitu tion, retaining 14-50% of the potency of ET. Ala analogues of the ET i someric disulfide arrangement (Cys(1,11) and Cys(3,15)) were always le ss active than the corresponding analogues with the native disulfide p airings (Cys(1,15) and Cys(3,11)).