ACTIVATION OF NEUROPEPTIDE Y1-RECEPTOR AND NEUROPEPTIDE Y2-RECEPTOR BY SUBSTITUTED AND TRUNCATED NEUROPEPTIDE-Y ANALOGS - IDENTIFICATION OF SIGNAL EPITOPES

Neuropeptide Y (NPY-(1-36)) acts on Y1 and Y2 receptors at the sympathetic neuroeffector junction. Various truncated NPY analogs were tested in the isolated guinea-pig caval vein where NPY is a vasoconstrictor (Y1 receptors) and in isolated rat vas deferens, by monitoring the suppression of electrically evoked contractions (Y2 receptors). The aim of this study was to define which parts of the NPY-(1-36) molecule were required to activate these receptors. NPY-(1-36), [Pro34]NPY and [Glu16,Ser18,Ala22,Leu28,31] NPY (ESALL-NPY), the latter being an analog with increased alpha-helicity in the 14-31 region, evoked vasoconstriction with similar potency and efficacy. Cyclic as well as linear NPY analogs having the 4 to 7 N-terminal amino acid residues linked to the 9 to 19 C-terminal residues by an 8-aminooctanoic acid (Aoc) residue were 25-50 times less potent than NPY-(1-36) itself. In the cyclic analogs, a disulfide bond was introduced to bring the N- and C-termini close together. Linear Aoc 2-27 -NPY was virtually inactive. The Y1 receptor needs an intact N-terminal end of NPY in order to become fully activated. The requirements for the C-terminus are less stringent, since substitutions in this part of the molecule resulted in fully active analogs. The central portion of the molecule may impose steric constraints on the N- and C-terminal ends, thereby facilitating Y1 receptor activation, but it does not seem to be essential for receptor recognition. NPY-(2-36) and NPY-(5-36) were only slightly less potent than the parent molecule in suppressing electrically evoked twitches in the vas deferens. ESALL-NPY was virtually equipotent with NPY-(1-36). [Aoc8-17]NPY and [Aoc5-24]NPY and the corresponding cyclic analogs effectively suppressed the stimulated twitches, but were about 10 times less potent than NPY-(1-36). [Aoc2-27]NPY was much less potent than the parent molecule. Substitution with proline in position 34 resulted in a marked loss of efficacy and potency. The results show that extensive N-terminal or central truncation does not greatly impair Y2 receptor recognition, whereas NPY analogs with proline in position 34 are poorly recognized. Hence, the Y2 receptor requires an intact NPY C-terminus, whereas demands for the N-terminal and mid-molecule parts are less stringent.