STRUCTURAL REQUIREMENTS FOR THE OCCUPANCY OF RAT-BRAIN PACAP RECEPTORS AND ADENYLATE-CYCLASE ACTIVATION

N-terminally shortened analogues of PACAP(1-27) and PACAP(1-38), and a nalogues modified in position 1, 2 or 3 were compared for their abilit y to interact with PACAP receptors and to activate or inhibit adenylat e cyclase in rat brain hippocampus membranes. In the PACAP(1-27) serie s, deletion of the first two amino acids decreased the potency 3000-fo ld. PACAP fragments (3-27) to (9-27) were inactive on the enzyme. N-te rminally shortened PACAP(1-38) analogues showed a similar profile but were 70 to 300-fold more potent than their PACAP(1-27) equivalents. PA CAP(6-27) and PACAP(6-38) were competitive inhibitors of the PACAP(1-2 7) stimulated enzyme. The K-d values of PACAP(6-27) and PACAP(6-38) we re of 1000 and 2 nM respectively. Surprisingly, the K-d values of PACA P(6-31) and (6-35), that were also unable to stimulate adenylate cycla se activity, were of 3 and 300 nM respectively. Replacement of His 1 b y Phe1 in PACAP(1-27) reduced the potency 600-fold. Replacement of Ser 2 by Ala2 in PACAP(1-27) and PACAP(1-38) was of little consequence. Su bstitution of Ser2 by Phe2, DPheZ, Arg2 or DArg2 reduced 60 to 1000-fo ld the PACAP(1-27) potency but only 7 to 30-fold the PACAP(1-38) poten cy. Phe2 derivatives were inactive on the enzyme. Replacement of Asp3 by Asn reduced 4000-fold the PACAP(1-27) potency. These results sugges t that three domains of PACAP(1-38) were involved in binding to rat br ain receptors: the N-terminal triplet His-Ser-Asp, the (11-27) area an d the C-terminal (27-38) extension; the integrity of two out of three domains sufficed for high affinity interaction at the binding level wh ile His1 and Ser2 and part of the 11-27 area were key elements in rece ptor-effector coupling.