Pharmacological characterization of cloned human NK-2 (neurokinin A) receptor expressed in a baculovirus/Sf-21 insect cell system.
Using the novel ligand [4,5-3H-Leu9]neurokinin A ([4,5-3H-Leu9] NKA) in a receptor binding assay, we characterized the pharmacology of a cloned 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 couple expression to the polyhedrin promoter, and the recombinant baculovirus was isolated and used to infect Sf-21 insect cells. hNK-2R expression levels were monitored by Northern blots and 125-I-NKA binding assays. Isolates demonstrating the highest specific binding of 125-I-NKA were grown and membrane preparations from high-speed centrifugations were prepared from both hNK-2R-expressing and wild-type virus-infected cells. [3H]NKA bound in a protein-dependent, saturable (Bmax = 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). [3H]NKA binding was rapid (k1 = 0.085 nM-1 x min-1) and reversible (t1/2 = 4-5 min). Equilibrium binding experiments demonstrated binding to a mixture of receptors in high and low affinity states (Kd1 = 2.28 +/- 0.26 nM and Kd2 = 266 +/- 91 nM). Binding to hNK-2R was greatly enhanced (400%-600%) by Ca2+ and Mg2+ (EC50 values of 30 microM and 140 microM, respectively), whereas guanosine-5'-O-(3'-thio)triphosphate and guanosine-5'-(beta, gamma-imido)diphosphate were inhibitory. Competition experiments with agonists also demonstrated binding to high and low affinity states, with the following order of potency: NKA > [Nle10]NKA(4-10) > [beta-Ala8]NKA(4-10) >> substance P; Senktide and the NK-1 antagonist CP96,345 (10 microM) did not inhibit binding. Inhibition of binding by selective NK-2 antagonists was consistent with a single affinity state and demonstrated the following order of affinity: SR48,968 >> MEN10,376 > 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, with pharmacological selectivity compatible with the NK-2A receptor subtype.
Department of Pharmacology, ZENECA Pharmaceuticals Group, a Business Unit of ZENECA Inc., Wilmington, Delaware 19897.
D
D
Aharony
J
J
Little
S
S
Powell
B
B
Hopkins
K R
KR
Bundell
W L
WL
McPheat
R D
RD
Gordon
G
G
Hassall
R
R
Hockney
R
R
Griffin
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001549:Benzamides / Q000494:pharmacology; D001665:Binding Sites; D015152:Blotting, Northern; D002460:Cell Line; D003001:Cloning, Molecular; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009036:Moths; D015288:Neurokinin A / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D010880:Piperidines / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011869:Radioligand Assay; D018041:Receptors, Neurokinin-2; D017981:Receptors, Neurotransmitter / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D001549:Benzamides; D010446:Peptide Fragments; D010880:Piperidines; D018041:Receptors, Neurokinin-2; D017981:Receptors, Neurotransmitter; D011994:Recombinant Proteins; C069192:neurokinin A(4-10), Tyr(5)-Trp(6,8,9)-Lys(10)-; C073839:SR 48968; D015288:Neurokinin A; D019204:GTP-Binding Proteins