PHARMACOLOGICAL CHARACTERIZATION OF CLONED HUMAN NK-2 (NEUROKININ-A) RECEPTOR EXPRESSED IN A BACULOVIRUS SF-21 INSECT-CELL SYSTEM/

Using the novel ligand [4,5-H-3-Leu9]neurokinin A ([4,5-H-3-Leu9] NKA) in a receptor binding assay, we characterized the pharmacology of a c loned neurokinin NK-2 receptor from human lung (hNK-2R), expressed in baculovirus-infected Sf-21 insect cells. Functional hNK-2R cDNA clones were isolated from human lung using a polymerase chain reaction-based methodology. hNK-2R was cloned into pAcYM1, a vector designed to coup le expression to the polyhedrin promoter, and the recombinant baculovi rus was isolated and used to infect Sf-21 insect cells. hNK-2R express ion levels were monitored by Northern blots and I-125-NKA binding assa ys. Isolates demonstrating the highest specific binding of I-125-NKA w ere grown and membrane preparations from high-speed centrifugations we re prepared from both hNK-2R-expressing and wild-type virus-infected c ells. [H-3]NKA bound in a protein-dependent, saturable (B(max) = 820 /- 167 fmol/mg of protein), and highly specific (88 +/- 5%) manner to hNK-2R, but not to membranes from cells infected with wild-type virus (14 +/-8%, 7 +/- 10 fmol/mg of protein). [H-3]NKA binding was rapid (k 1 = 0.085 nm-1.min-1) and reversible (t1/2 = 4-5 min). Equilibrium bin ding experiments demonstrated binding to a mixture of receptors in hig h and low affinity states (K(d1) = 2.28 +/- 0.26 nm and K(d2) = 266 +/ - 91 nm). Binding to hNK-2R was greatly enhanced (400%-600%) by Ca2+ a nd Mg2+ (EC50 values of 30 muM and 140 mum, respectively), whereas gua nosine-5'-O-(3'-thio)triphosphate and guanosine-5'-(beta,gamma-imido)d iphosphate were inhibitory. Competition experiments with agonists also demonstrated binding to high and low affinity states, with the follow ing order of potency: NKA > [Nle10]NKA(4-10) > [beta-Ala8]NKA(4-10) mu ch greater than substance P; Senktide and the NK-1 antagonist CP96,345 (10 muM) did not inhibit binding. Inhibition of binding by selective NK-2 antagonists was consistent with a single affinity state and demon strated the following order of affinity: SR48,968 much greater than ME N10,376 > L659,877 > R396. These data suggest that infection of Sf-21 cells with baculovirus expression vector harboring the cDNA of hNK-2R resulted in expression of high affinity, G protein-coupled hNK-2R, wit h pharmacological selectivity compatible with the NK-2A receptor subty pe.