Mechanisms of constitutive and agonist-induced 5-HT2B internalization, persistent endosomal signaling and paradoxical regulation of agonist pharmacology

Abstract

Certain ergot derivatives, particularly cabergoline, produce wash-resistant signaling through the 5-HT_2B receptor persisting for many hours without loss of potency or efficacy. Previously, we reported that this signaling may be mediated by sequestered or internalized receptors. Here, we evaluated numerous mechanistic aspects of 5-HT_2B internalization and wash-resistant signaling and directly addressed the role of internalization. In the absence of an agonist, 5-HT_2B undergoes robust, constitutive internalization and recycling and is distributed at equilibrium between cell surface and intracellular compartments. Both constitutive and agonist-induced internalization are mediated through dynamin-dependent clathrin-mediated endocytosis. Constitutive internalization is unaffected by application of 5-HT_2B inverse agonists. We identified two, adjacent di-leucine motifs followed by a di-acidic cluster in the C-terminal tail of 5-HT_2B that are responsible for constitutive internalization of the receptor. Mutations in either of the leucine clusters or in the di-acidic motif partially inhibit constitutive 5-HT_2B internalization. A 5-HT_2B mutant in which both di-leucine clusters are disrupted, displays no constitutive internalization while undergoing robust agonist induced internalization. We demonstrate that wash-resistant signaling of ergots is mediated by persistently/irreversibly internalized signaling receptor complexes. Paradoxically, the potencies of ergot agonists are influenced by receptor internalization; measured potencies are reduced upon inhibition of receptor internalization, while potencies for 5-HT or other conventional agonists are unaffected. This phenomenon represents a novel mechanism by which agonist-dependent kinetics of receptor internalization and recycling affects not only the duration of receptor signaling, but also a basic pharmacological parameter such as agonist potency.