A Novel Mechanism of Neurokinin-1 Receptor Resensitization

Abstract

Prolonged or repeated activation of many G protein-coupled receptors induces rapid desensitization followed by a period during which receptors are resensitized. In this study, concanavalin A (Con A) and monensin were used to investigate the mechanisms of desensitization and resensitization of the neurokinin-1 receptor. Con A inhibits internalization, whereas monensin prevents receptor recycling. The effects of Con A and monensin on desensitization, resensitization, receptor phosphorylation, endocytosis, and recycling of the neurokinin-1 receptor were assessed. Desensitization was defined as the decrease in the ability of substance P (SP) to elicit an intracellular Ca 2ϩ response after a prolonged SP exposure. Resensitization was characterized as the return of SP responsiveness. Under control conditions, desensitization occurred after a 5-min exposure to agonist. Resensitization was evident 30 min after agonist washout. Neither monensin nor Con A prevented desensitization. Monensin completely inhibited resensitization, whereas Con A decreased but did not completely block resensitization. Receptor phosphorylation was increased after agonist activation and returned to basal levels after a recovery period. Neither Con A nor monensin altered the amount of agonist-specific receptor phosphorylation. Receptor binding analysis showed that plasma membrane receptors were internalized after a 5-min agonist exposure. Receptor recycling was not observed after a 1-h recovery period; however, resensitization was apparent. Taken together, these results suggest that rapid neurokinin-1 receptor desensitization can occur without receptor internalization and that resensitization occurs before receptor recycling.

Phosphorylation of a Carboxyl-terminal Serine within the κ-Opioid Receptor Produces Desensitization and Internalization

“…These results may suggest that alternative mechanisms to receptor internalization exist in the process of receptor resensitization. Indeed, a recent study with desensitized neurokinin-1 receptors identified a receptor resensitization that occurred before receptor recycling (34), consistent with the findings described here.…”

Neurokinin-1 Receptor Resensitization Precedes Receptor Recycling

“…Thus, the cells experienced four solution changes over a period of at least 30 min before the radioligand was added. Since the dissociation rate constant of substance P is 0.28 min Ϫ1 (Takeda et al,1992), the unlabeled substance P should have been removed during the washes, and the solution changes prior to the addition of radiolabeled substance P. We confirmed the effectiveness of these steps to remove unlabeled substance P in previous experiments (Bennett et al, 2002).…”

“…While studying the effects of monensin and concanavalin A on rNK1R desensitization, internalization, recycling, and resensitization, we found that, following agonist-induced rNK1R desensitization and internalization, there was an increase in rNK1R-mediated Ca 2ϩ signaling following a 60-min recovery period without agonist, but there was not an equivalent return of plasma membrane receptor binding during the same recovery period (Bennett et al, 2002). Using a fluorescent substance P analog, we also showed that internalization corresponds with a decrease in plasma membrane binding of radiolabeled substance P following receptor activation, and recycling corresponds with an increase in binding following internalization.…”

“…Following activation of the NK1R, the receptor undergoes rapid desensitization, and the receptors are internalized within minutes (Garland et al, 1996;Bennett et al, 2002). Phosphorylation and G protein uncoupling are thought to mediate desensitization and internalization of most GPCRs (reviewed by Ferguson, 2001).…”

Separation of μ-Opioid Receptor Desensitization and Internalization: Endogenous Receptors in Primary Neuronal Cultures

“…This sequence could result in a blockade of signaling without internalization, however, the binding of arrestin to the ligand/receptor complex is thought to proceed to endocytosis via clathrin-coated pits (Law and Loh, 1999;Ferguson, 2001;von Zastrow et al, 2003). The separation of internalization and desensitization has been reported for other G-protein-coupled receptors such as ␤ 2 -adrenergic and neurokinin-1 receptors (Pippig et al, 1995;Bennett et al, 2002), but it has also been show that the desensitization of somatostatin and V1b vasopressin receptors requires internalization (Beaumont et al, 1998;Hassan and Mason, 2005). Although this sequence has been established in many heterologous systems, the role of this process in MOR signaling is the subject of some debate.…”