Characterization of a novel VPAC1 selective agonist and identification of the receptor domains implicated in the carboxyl-terminal peptide recognition

Vasoactive Intestinal Polypeptide (VIP) interacts with a high affinity to two subclasses of G protein coupled receptors named VPAC1 and VPAC2, and has a 3–10 fold preference for VPAC1 over VPAC2 receptors. Selective ligands for each receptor subclass were recently described. [R16]-PACAP (1–23) and [L22]-VIP are two selective VPAC1 agonists. Chimaeric human VPAC2-VPAC1 recombinant receptors expressed in CHO cells were used to identify the receptor domains implicated in these two selective ligands recognition. The VPAC2 preference for [R16]-PACAP (1–27) over [R16]-PACAP (1–23) did not require the receptor's NH2-terminus domain but involved the whole transmembrane domain. In contrast, the selectivity of [L22]-VIP depended only on the presence of the NH2 terminus and EC2 domains of the VPAC1 receptor. The present data support the idea that in the GPCR-B family of receptors the different selective ligands require different domains for their selectivity, and that the peptides carboxyl terminal sequence (amino acids 24–27) folds back on the transmembrane receptor domain, close to the peptides, aminoterminus. Keywords: Vasoactive intestinal peptide, VPAC1 receptors, VPAC2 receptors, VPAC1-selective agonists, peptide binding domains Introduction Vasoactive intestinal polypeptide (VIP) interacts with two receptor subtypes, previously named VIP1 and VIP2 and currently known as VPAC1 and VPAC2 (Harmar et al., 1998), that have been cloned in rat (Lutz et al., 1993; Ishihara et al., 1992), human (Couvineau et al., 1994; Svoboda et al., 1994) and mouse (Inagaki et al., 1994). These receptors belong to the class B of the G-protein coupled receptors that includes the calcitonin-, the corticotrophin releasing factor-, an insect diuretic hormone-, the gastric inhibitory peptide-, the glucagon-, the glucagon-like peptide I-, the growth hormone releasing factor-, the parathyroid hormone-, the pituitary adenylate cyclase activating polypeptide (PACAP)-, the secretin-, the VPAC1- and VPAC2- and also orphan receptors (Donnelly, 1997). All these receptors have significant sequence homology (especially in the transmembrane domains) and possess a large extracellular NH2-terminal domain with six conserved cysteine residues. They are all positively coupled to adenylate cyclase but some of them may also be coupled to other effectors (Van Rampelbergh et al., 1997; Spengler et al., 1993). In situ hybridization reveals that both VIP receptor subtypes are transcribed – and thus probably expressed – in different tissues or cells (Usdin et al., 1994). This is particularly obvious in brain where their localization in many areas appear mutually exclusive (Vertongen et al., 1997). The development of agonists and antagonists selective for each receptor subclass is therefore of great pharmacological importance. The neuropeptides VIP and PACAP recognize both receptor subtypes with a high affinity (Couvineau et al., 1996; Svoboda et al., 1994). The peptide with a NH2-terminal histidine and a COOH-terminal isoleucine amide (PHI) or its longer form with a COOH-terminal valine amide (PHV) – that are co-synthesized and released with VIP – have a higher affinity for the human VPAC2 than for the human VPAC1 receptor (Couvineau et al., 1996; Gourlet et al., 1998), which is not the case for the rat receptors (Couvineau et al., 1996). Several synthetic ligands, selective for each receptor subtype have been described recently (Gourlet et al., 1997a,1997b; 1998; Xia et al., 1997). Amongst them, [R16]-PACAP (1–23) (as example of truncated peptide) and [L22]-VIP (as example of single change in the peptide sequence) are selective VPAC1 receptor agonists (this study and Gourlet et al., 1998). Little is known to date about the receptor determinants that are responsible for the different selectivity profiles observed for these related receptors. In the present study, we constructed and stably expressed in Chinese hamster ovary (CHO) cells chimaeric receptors made by the replacement of parts of the human VPAC1 receptor sequence by the corresponding sequence of the human VPAC2 receptor to identify the receptor domains involved in this selectivity. The shortened original PACAP (1–27) derivative, [R16]-PACAP (1–23) and a mono-substituted VIP analogue [L22]-VIP were used in this study as VPAC1 selective ligands and were compared to [R16]-PACAP (1–27) and VIP respectively.