RRC ID 38947
Author Ogo H, Kuroyanagi N, Inoue A, Nishio H, Hirai Y, Akiyama M, DiMaggio DA, Krause JE, Nakata Y.
Title Human astrocytoma cells (U-87 MG) exhibit a specific substance P binding site with the characteristics of an NK-1 receptor.
Journal J Neurochem
Abstract To investigate substance P (SP) receptors on an established human astrocytoma cell line (U-87 MG), [3H][Sar9,Met(O2)11]-SP, a selective SP receptor agonist, was used to identify and characterize the cell membrane binding sites for SP. SP receptor mRNA was examined by solution hybridization analysis, and the existence of SP binding protein on the surface of membranes was evaluated by flow cytometry using an anti-SP binding protein antibody. In U-87 MG and U-373 MG RNA preparations, transcripts were identified that corresponded to both mature and partially spliced receptor forms. In U-87 MG cell membrane-enriched preparations, the binding of [3H][Sar9,Met(O2)11]-SP was found to be time and cell number dependent, specific, saturable, and of high affinity. Equilibrium binding analysis revealed a single class of binding sites with an apparent KD of 1.15 +/- 0.15 nM and a Bmax of 108 +/- 9.8 fmol/mg of protein. [3H][Sar9, Met(O2)11]-SP binding was basically not influenced by addition of mono (Na+, Li+) or divalent (Mg2+, Mn2+, Ca2+) cations; only high doses of divalent cations decreased the binding. GTP and guanylyl-5'-imidodiphosphate, but not GDP and GMP, reduced the Bmax without changing the affinity of [3H][Sar9,Met(O2)11]-SP. We also examined the effects of pretreatment with three lectins [concanavalin A (con A), wheat germ agglutinin (WGA), and Lens culinaris agglutinin (LCA)] to determine the nature of carbohydrate chains on the U-87 MG cell. Of three lectins analyzed for effects on agonist binding, WGA and LCA had an inhibitory effect, whereas con A was ineffective. These results suggest that SP receptors on the human astrocytoma cell line U-87 MG have either a biantennary complex-type or a high mannose-type of carbohydrate chain and may be regulated by GTP-binding protein(s).
Volume 67(5)
Pages 1813-20
Published 1996-11-1
DOI 10.1046/j.1471-4159.1996.67051813.x
PMID 8863485
MeSH Astrocytoma Binding Sites Binding, Competitive Cations, Monovalent / pharmacology Cell Line Exons Glioblastoma Guanine Nucleotides / pharmacology Guanosine Triphosphate / pharmacology Humans Kinetics Lectins / pharmacology RNA, Antisense RNA, Complementary RNA, Messenger / metabolism Receptors, Neurokinin-1 / agonists Receptors, Neurokinin-1 / biosynthesis Receptors, Neurokinin-1 / metabolism* Substance P / analogs & derivatives Substance P / metabolism* Tritium Tumor Cells, Cultured
IF 4.066
Times Cited 12
WOS Category NEUROSCIENCES BIOCHEMISTRY & MOLECULAR BIOLOGY
Resource
Human and Animal Cells U-87 MG(RCB0419)