Molecular cloning and functional expression of a VIP-specific receptor

Abstract

Three receptors for VIP and pituitary adenylate cyclase-activating peptide (PACAP) have been cloned and characterized: PAC₁, with high affinity for PACAP, and VPAC₁ and VPAC₂ with equally high affinity for VIP and PACAP. The existence of a VIP-specific receptor (VIP_s) in guinea pig (GP) teniae coli smooth muscle was previously surmised on the basis of functional studies, and its existence was confirmed by cloning of a partial NH₂-terminal sequence. Here we report the cloning of the full-length cDNAs of two receptors, a VPAC₂ receptor from GP gastric smooth muscle and VIP_s from GP teniae coli smooth muscle. The cDNA sequence of the VIP_s encodes a 437-amino acid protein ( M_r 49,560) that possesses 87% similarity to VPAC₂ receptors in rat and mouse and differs from the VPAC₂ receptor in GP gastric smooth muscle by only two amino-acid residues, F⁴⁰F⁴¹ in lieu of L⁴⁰L⁴¹. In COS-1 cells transfected with the GP teniae coli smooth muscle receptor, only VIP bound with high affinity (IC₅₀ 1.4 nM) and stimulated cAMP formation with high potency (EC₅₀ 1 nM). In contrast, in COS-1 cells transfected with the GP gastric smooth muscle receptor, both VIP and PACAP bound with equally high affinity (IC₅₀ 2.3 nM) and stimulated cAMP with equally high potency (EC₅₀ 1.5 nM). We conclude that the receptor cloned from GP teniae coli smooth muscle is a VIP_s distinct from VPAC₁ and VPAC₂ receptors. The ligand specificity in this species is determined by a pair of adjacent phenylalanine residues (L⁴⁰L⁴¹) in the NH₂-terminal ligand-binding domain.