Vol. 281, Issue 2, 629-633, 1997

Novel Cyclic Peptide Agonist of High Potency and Selectivity for the Type II Vasoactive Intestinal Peptide Receptor¹

Menghang Xia, Sunil P. Sreedharan, David R. Bolin, Gary O. Gaufo and Edward J. Goetzl

Departments of Medicine and Microbiology-Immunology, University of California, San Francisco, California (M.X., S.P.S., G.O.G., E.J.G), and Department of Molecular Sciences, Hoffmann-La Roche, Inc., Nutley, New Jersey (D.R.B.)

Ro 25-1392 [Ac-Glu⁸,OCH₃-Tyr¹⁰,Lys¹²,Nle¹⁷,Ala¹⁹,Asp²⁵,Leu²⁶,- Lys^27,28-vasoactive intestinal peptide(cyclo 21-25)] is a cyclic peptide analog of vasoactive intestinal peptide (VIP) that potently exerts cellular effects typical of VIP. The selectivity of Ro 25-1392 for type I (VIPR1) and type II (VIPR2) VIP receptors was investigated first in competitive binding studies using Chinese hamster ovary cell transfectants stably expressing recombinant human VIPR1 and VIPR2. Nonradioactive Ro 25-1392 was as potent a competitive inhibitor as VIP for the binding of ¹²⁵I-VIP to VIPR2 transfectants (K_i = 9.6 ± 1.0 and 16 ± 1.7 nM, respectively; mean ± S.E.M., n = 4). In contrast, Ro 25-1392 had a very low affinity for VIPR1, compared with VIP, and attained a maximum of only 40% mean inhibition of binding of ¹²⁵I-VIP at 1 µM. The affinity of VIP (K_i = 3.4 ± 1.5 nM, mean ± S.E.M., n = 4) for binding to VIPR1 was 1000-fold greater than that of Ro 25-1392. Ro 25-1392 evoked concurrent and concentration-dependent increases in intracellular levels of calcium and cyclic AMP (EC₅₀ = 3.0 ± 0.4 nM, mean ± S.E.M., n = 4) in VIPR2 transfectants, but not in VIPR1 transfectants. The VIP receptor specificity of Ro 25-1392 was confirmed by preincubation of Chinese hamster ovary transfectants with 0.1 µM Ro 25-1392 for 18 hr at 37°C, to down-regulate each type of VIP receptor. Pretreatment of VIPR2 transfectants with Ro 25-1392 decreased B_max by a mean of 58% and VIP-induced increases in the intracellular concentration of cyclic AMP by a mean of 65%. In contrast, there was no significant change in VIPR1 transfectants after pretreatment with Ro 25-1392. Ro 25-1392 thus is selectively recognized by VIPR2, with consequent initiation of cyclic AMP and Ca⁺⁺ signals and down-regulation of VIPR2. This potent analog of VIP may prove useful for investigations of VIPR2-mediated physiological effects of VIP and exploration of the roles of VIPR2 in diseases.