Vasoactive Intestinal Peptide (VIP)1 Receptor

Abstract

Vasoactive intestinal peptide (VIP)1 receptors in rats and humans recognize peptide histidine isoleucineamide (PHI) with high and low affinity, respectively. We took advantage of this phenotypic difference to identify the domain responsible for the selective recognition of PHI by rat and human receptors which display >80% sequence identity. After transfection of human and rat receptors in COS cells, the ratio of IC50 for PHI/IC50 for VIP (referred to as P/V) in inhibiting 125I-VIP binding was shown to be >1,000 and <40, respectively. Construction of eight rat/human receptor chimerae by overlap polymerase chain reaction and determination of their P/V ratios demonstrated that the critical domain for PHI recognition is present within a sequence comprising part of the first extracellular loop and third transmembrane domain. This domain contains three different amino acids numbered according to human and rat sequences, respectively, e.g. Gln207 (human) versus His208 (rat), Gly211 versus Ala212 and Met219 versus Val220. Site-directed mutagenesis introducing individual, double, or triple mutations in a chimeric construct revealed that all three amino acids were involved in the recognition of PHI. Triple mutations were then introduced in the wild-type receptors i.e. Q207H, G211A, M219V human VIP1 receptor and H208Q, A212G, V220M rat VIP1 receptor, resulting in a complete change in their phenotype from human to rat and from rat to human, respectively. The results demonstrate that three nonadjacent amino acids are responsible for the selective recognition of PHI by human and rat VIP1 receptors.

Diffuse Pharmacophoric Domains of Vasoactive Intestinal Peptide (VIP) and Further Insights into the Interaction of VIP with the N-terminal Ectodomain of Human VPAC1 Receptor by Photoaffinity Labeling with [Bpa6]-VIP

“…Five other mutants were also constructed in which residues within the 104 -108 receptor sequence were individually replaced by alanine. Oligonucleotide-directed mutagenesis was performed as previously described (4). Identification of the desired mutations was obtained by direct sequencing of the mutated region (4).…”

“…Identification of the desired mutations was obtained by direct sequencing of the mutated region (4). The mutants were stably transfected in CHO cells as described (4).…”

“…3). These studies made it possible to delineate the receptor domains involved in high affinity VIP binding (3), selectivity toward some natural peptide agonists (4,5) and also activation of adenylyl cyclase (6). With respect to VIP binding, it appeared that the N-terminal ectodomain of the receptor plays a crucial role, although it is not sufficient to ensure high affinity (3).…”

Identification of Key Residues for Interaction of Vasoactive Intestinal Peptide with Human VPAC1 and VPAC2Receptors and Development of a Highly Selective VPAC1Receptor Agonist

“…Although the structure-function relationship of VIP receptors, including VPAC 1 and VPAC 2 , has been recently documented (2,9,(12)(13)(14)(15)(16)(17)(18)(19)(20), the structure-function relationship of VIP itself is still poorly understood. Some old studies carried out before the characterization and cloning of VIP receptor subtypes (21)(22)(23) indicated that: (i) the entire sequence of VIP is required for full biological function (21,22); (ii) VIP-related peptides having significant sequence homologies with VIP such as peptide histidine methionine amide, secretin, GRF, and helodermin behave as low potency VIP agonists (23); (iii) there are important differences between species in the pharmacology of VIP receptors, especially between rodents and humans (2,15).…”

“…This subfamily referred to as class II (2) also comprises receptors for parathyroid hormone, calcitonin, corticotropin-releasing factor, and the so called EGF-TM7 receptors (11). Class II family of receptors for peptides display several common properties including large N-terminal extracellular domains containing highly conserved cystein residues, N-terminal leader sequences, and complex gene organization with many introns (2).Although the structure-function relationship of VIP receptors, including VPAC 1 and VPAC 2 , has been recently documented (2,9,(12)(13)(14)(15)(16)(17)(18)(19)(20), the structure-function relationship of VIP itself is still poorly understood. Some old studies carried out before the characterization and cloning of VIP receptor subtypes (21-23) indicated that: (i) the entire sequence of VIP is required for full biological function (21, 22); (ii) VIP-related peptides having significant sequence homologies with VIP such as peptide histidine methionine amide, secretin, GRF, and helodermin behave as low potency VIP agonists (23); (iii) there are important differences between species in the pharmacology of VIP receptors, especially between rodents and humans (2, * The costs of publication of this article were defrayed in part by the payment of page charges.…”