Molecular Pharmacology Volume 60, Issue 3, September 2001, Pages 534-540
Neuropeptide Y (NPY) binds to a family of G-protein coupled receptors termed Y 1, Y 2, Y 3, Y 4, Y 5, and y 6. The use of various receptor subtype-selective agonists and antagonists has facilitated identification of the receptor subtypes responsible for mediating many of the biological effects of NPY. For example, the potent orexigenic activity of NPY is believed to be mediated by both the Y 1 and Y 5 receptor subtypes. Several selective Y 5 receptor agonists that stimulate food intake in rodents are available, but no selective Y 1 receptor agonist has been reported. We have identified several NPY analogs that bind the NPY Y 1 receptor with high affinity and exhibit full agonist activity, measured as inhibition of forskolin-stimulated cAMP production in cells expressing the cloned NPY Y 1 receptor. d-Arg 25-NPY, d-His 26-NPY, Des-AA 10–17Cys 7,21,Pro 34-NPY, Des-AA 11–18Cys 7,21,d-Lys 9(Ac)-NPY, Des-AA 11–18Cys 7,21,d-Lys 9(Ac),Pro 34-NPY, Des-AA 11–18Cys 7,21,d-Lys 9(Ac),d-His 26-NPY and Des-AA 11–18Cys 7,21,d-Lys 9(Ac),d-His 26, Pro 34-NPY bind the NPY Y 1 receptor with K i values of 0.9 ± 0.2, 2.0 ± 0.3, 0.2 ± 0.05, 0.7 ± 0.1, 0.2 ± 0.01, 2.2 ± 0.3, and 1.2 ± 0.3 nM, respectively, and inhibit forskolin-stimulated cAMP production with EC 50 values of 0.2 ± 0.02, 0.5 ± 0.04, 0.3 ± 0.03, 0.5 ± 0.05, 0.4 ± 0.16, 5.3 ± 0.32, and 5.1 ± 0.97 nM, respectively. These peptides are highly selective for the NPY Y 1 receptor relative to the NPY Y 2, Y 4, and Y 5 receptors. d-Arg 25-NPY, d-His 26-NPY and Des-AA 11–18Cys 7,21, d-Lys 9(Ac),d-His 26,Pro 34-NPY stimulate food intake dose-responsively in Long-Evans rats for at least 4 h after intracerebroventricular administration. Although the involvement of Y 1 receptors in several physiological activities, such as vasoconstriction and anxiolysis, remains to be investigated, adequate tools are now available.
Peptides were assembled by solid phase peptide synthesis techniques using the tertiary-butyloxycarbonyl strategy. Peptides were purified by preparative high-performance liquid chromatography and characterized using capillary zone electrophoresis, mass spectrometry, and analytical high-performance liquid chromatography as described previously (Kirby et al., 1993b).
Cloning and expression of the human and rat NPY Y 1, Y 2, Y 4, and Y 5 receptors and the construction of a chimeric rat/human NPY Y 5
Two Y 1-selective NPY analogs were identified from the complete series of d-amino acid substituted peptides, d-Arg 25-NPY (1) and d-His 26-NPY (2). 1 binds the Y 1 receptor with 12-, 82-, and 48-fold higher affinity than the Y 2, Y 4, and Y 5 receptor subtypes, respectively. Similarly, the affinity of 2 for the Y 1 receptor is 14-, 10-, and 17-fold greater than for the Y 2, Y 4, and Y 5 receptor subtypes, respectively. The affinity of 1 for the NPY Y 1 receptor is 3.2 times less than that of NPY,
The study of the physiological responses mediated by each NPY receptor subtype has been facilitated by the identification of subtype-selective agonists. Agonists selective for the Y 2 receptor C2-NPY (McLean et al., 1990), the Y 4 receptor rat PP (Bard et al., 1995), and the Y 5 receptor {DTrp 32NPY (Balasubramaniam et al., 1994), DTrp 34NPY (Parker et al., 2000), Ala 31,Aib 32NPY, hPP 1–17,Ala 31,Aib 32hNPY, and cPP 1–7,NPY 19–23,Ala 31, Aib 32,Gln 34hPP (Cabrele et al., 2000)} have been
