Abstract

Clone N1E-115 cells have specific, high-affinity binding sites for neurotensin (NT). These receptors mediate formation of cyclic GMP and other second messengers. We studied the effects of extended exposure of cells to [D-Lys8]NT(8-13) (NT2), a peptidase-resistant NT analog, on NT's ability to stimulate cyclic GMP synthesis and to bind [3H]NT to its sites on these cells. When intact N1E-115 cells were preincubated with NT2 (1 microM) for various times (15 min to 12 hr) at 37 degrees C, maximal desensitization and binding site (Bmax) loss occurred after only 15 min. Receptor affinity for [3H]NT did not change. At 0 degrees C NT2 for 15 min caused no down-regulation. After 15 min of preincubation with NT2 at 37 degrees C, the recovery of receptor binding and function after a 10 to 20 min lag-time was rapid (T1/2 = 15 min and 19 min, for binding and cyclic GMP response, respectively). After 12 hr of NT2 preincubation, it was slow (T1/2 = 212 min). Incubation of cells with cycloheximide (70 microM) for 5 hr after their exposure to NT2 for 15 min did not change the rate or extent of recovery of NT Bmax. However, cycloheximide did decrease the recovery of NT Bmax after exposure of cells to NT2 for 12 hr. Cycloheximide alone did not change NT Bmax. These data suggest that this decrease of NT receptor binding after short (15 min) and long (12 hr) exposure times to agonist involves two consecutive steps: intracellular sequestration of recyclable NT receptors, followed by receptor degradation causing true down-regulation.