HUMAN ASTROCYTOMA-CELLS (U-87 MG) EXHIBIT A SPECIFIC SUBSTANCE-P BINDING-SITE WITH THE CHARACTERISTICS OF AN NK-1 RECEPTOR

To investigate substance P (SP) receptors on an established human astr ocytoma cell line (U-87 MG), [H-3] [Sar(9), Met(O-2)(11)]-SP, a select ive SP receptor agonist, was used to identify and characterize the cel l membrane binding sites for SP. SP receptor mRNA was examined by solu tion hybridization analysis, and the existence of SP binding protein o n the surface of membranes was evaluated by flow cytometry using an an ti-SP binding protein antibody. In U-87 MG and U-373 MG RNA preparatio ns, transcripts were identified that corresponded to both mature and p artially spliced receptor forms. In U-87 MG cell membrane-enriched pre parations, the binding of [H-3][Sar(9), Met(O-2)(11)]-SP was found to be time and cell number dependent, specific, saturable, and of high af finity. Equilibrium binding analysis revealed a single class of bindin g sites with an apparent K-D of 1.15+/-0.15 nM and a B-max of 108+/-9. 8 fmol/mg of protein, [H-3][Sar(9), Met(O-2)(11)]-SP binding was basic ally 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, re duced the B-max without changing the affinity of [H-3] [Sar(9), Met(O- 2)(11)]-SP. We also examined the effects of pretreatment with three le ctins [concanavalin A (con A), wheat germ agglutinin (WGA), and Lens c ulinaris agglutinin (LCA)I to determine the nature of carbohydrate cha ins on the U-87 MG cell. Of three lectins analyzed for effects on agon ist binding, WGA and LCA had an inhibitory effect, whereas con A was i neffective. These results suggest that SP receptors on the human astro cytoma cell line U-87 MG have either a biantennary complex-type or a h igh mannose-type of carbohydrate chain and may be regulated by GTP-bin ding protein(s).