Characterization of a tachykinin peptide NK2 receptor transfected into murine fibroblast B82 cells.
Membranes isolated from a murine fibroblast B82 cell line (SKLKB82#3) transfected with the bovine stomach cDNA pSKR56S exhibited binding of [His(125I)1]neurokinin A (125I-NKA) to a single population of sites with a Bmax of 147 fmol/mg of protein and a Kd of 0.59 nM. Control cell lines had little or no specific binding. The ligand binding in SKLKB82#3 cells was reversible and was inhibited by peptides in the potency rank of neuropeptide gamma greater than neuropeptide K greater than neurokinin A greater than [10-norleucine]neurokinin A-(4-10) greater than substance P much greater than senktide (succinyl-Asp-Phe-MePhe-Gly-Leu-Met-NH2). Specific binding was enhanced by Mn2+, Mg2+, and Ca2+ and was inhibited by guanine nucleotide analogues. Thus, SKLKB82#3 cells have been transfected with NK2 receptors that have become associated with an endogenous guanine nucleotide-binding protein. In comparison with membranes from the hamster urinary bladder, a tissue enriched in NK2 receptors, NK2 receptor antagonists displayed markedly different potencies, either more or less potent, in inhibiting specific binding in membranes of the transfected cells. Furthermore, inhibition of 125I-NKA binding by nucleotide analogues was markedly different in SKLKB82#3 cells compared with hamster bladder tissue. The different binding profile in the cells is not due to an artefact introduced during cDNA transfection because a similar profile was also observed in bovine stomach membranes. These results may indicate the existence of two distinct NK2 receptors.
van Giersbergen
P L
PL
Marion Merrell Dow Research Institute, Cincinnati, OH 45215.
Shatzer
S A
SA
Henderson
A K
AK
Lai
J
J
Nakanishi
S
S
Yamamura
H I
HI
Buck
S H
SH
D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002460:Cell Line; D006224:Cricetinae; D005347:Fibroblasts / Q000378:metabolism; D005753:Gastric Mucosa / Q000378:metabolism; D007700:Kinetics; D017981:Receptors, Neurotransmitter / Q000235:genetics / Q000378:metabolism*; D018039:Receptors, Tachykinin; D015320:Tachykinins / Q000378:metabolism*; D014162:Transfection; D001743:Urinary Bladder / Q000378:metabolism
D017981:Receptors, Neurotransmitter; D018039:Receptors, Tachykinin; D015320:Tachykinins