Isolation and Charactrization of Neurokinin a Receptor cDNAs from Guinea-Pig Lung and Rabbit Pulmonary Artery

Abstract

cDNA clones for NK-2 receptors (NK-2R) were isolated from guinea-pig lung (GP1) and rabbit pulmonary artery (Rpa) using a polymerase chain reaction based methodology. The GPI NK-2R consists of 402 amino acids and encodes a protein with a relative molecular mass of 45,097. The Rpa NK-2R consists of 384 amino acids and encodes a protein with a relative molecular mass of 43,169. The GPI and Rpa NK-2Rs share significant amino acid sequence homology amongst themselves (90.l%), as well as with human, bovine, hamster and rat NK-2 receptors.The two receptors were stably transfected into mouse erythroleukemia cells, high-speed membranes were prepared from induced cells and their pharmacological properties examined utilizing [₃H]-NKA in a receptor-binding assay. [³H]NKA bound to both NK-2Rs with high affinity (K_D = 2-7 nM) and saturable (B_max = 633 - 9000 fmol/mg protein) manner which was inhibited by GTP analogs. Competition experiments with agonists demonstrated identical order of potency in both NK-2Rs: NKA > [Nle^1O]NKA(4-l0) > [β-Ala⁸]NKA(4-10) ≫ Substance P ⋙ Senktide. Similarly, an identical profile for both receptors was observed with selective NK-2 antagonists: SR48,968 > MEN10.376 ≫ R396. The rank order of antagonist affinity is consistent with that in cloned human NK-2R and the observations of NK-2 receptor pharmacology in native human, guinea pig and rabbit tissues.