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.
Highlights
Vasoactive intestinal peptide (VIP)[1] receptors in rats and humans recognize peptide histidine isoleucineamide (PHI) with high and low affinity, respectively
Taking advantage of the clear-cut phenotypic difference between rat and human VIP1 receptor with respect to PHI binding (IC50 values are 19 Ϯ 4 and 1420 Ϯ 340 nM, respectively), we investigated its genotypic basis by constructing rat/human VIP1 receptor chimerae
In this paper we demonstrate that the different phenotype of human and rat VIP1 receptors with regard to PHI recognition is due to a genotypic change affecting three nonadjacent amino acid residues located in the first extracellular loop and third transmembrane domain
Summary
Materials—Restriction enzymes and culture medium were obtained from Life Technologies, Inc. (Cergy Pontoise, France). Chimeric receptors were constructed using the two-step overlap polymerase chain reaction approach (20) with oligonucleotides consisting of human and rat VIP1 receptor sequences specifying the splicing junction. Site-directed Mutagenesis—Mutations were introduced into wildtype human and rat receptors and the R/H3 chimera subcloned into the pALTER vector (Promega, France) as described (12–14). Ligand Binding Assay—Membranes (200 g of protein/ml) derived from transfected cells were incubated for 60 min at 30 °C in 20 mM HEPES buffer, pH 7.4, containing 2% (w/v) bovine serum albumin, 0.1% (w/v) bacitracin, 0.05 nM 125I-VIP in the presence of increasing concentrations of VIP, PHI, or other competing peptides. Transfected cells in 12-well trays were incubated in the presence or absence of VIP in 1 ml of phosphate-buffered saline containing 2% (w/v) bovine serum albumin, 0.1% (w/v) bacitracin, and 1 mM 3-isobutyl-1-methylxanthine for 30 min at 20 °C. At the end of the incubation, the medium was rapidly removed, cells were washed, and 1 ml of 1 M perchloric acid was added. cAMP was measured by radioimmunoassay as described elsewhere (19)
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