Activation of neuropeptide Y 1 and neuropeptide Y 2 receptors by substituted and truncated neuropeptide Y analogs: identification of signal epitopes

Abstract

Neuropeptide Y (NPY-(1–36)) acts on Y 1 and Y 2 receptors at the sympathetic neuroeffector junction. Various truncated NPY analogs were tested in the isolated guinea-pig caval vein where NPY is a vasoconstrictor (Y 1 receptors) and in isolated rat vas deferens, by monitoring the suppression of electrically evoked contractions (Y 2 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), [Pro 34]NPY and [Glu 16, Ser 18, Ala 22, Leu 28,31] NPY (ESALL-NPY), the latter being an analog with increased α-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 Y 1 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 Y 1 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). [Aoc 8–17]NPY and [Aoc 5–24]NPY and the corresponding cyclic analogs effectively suppressed the stimulated twitches, but were about 10 times less potent than NPY-(1–36). [Aoc 2–27]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 tha extensive N-terminal or central truncation does not greatly impair Y 2 receptor recognition, whereas NPY analogs with proline in position 34 are poorly recognized. Hence, the Y 2 receptor requires an intact NPY C-terminus, whereas demands for the N-terminal and mid-molecule parts are less stringent.