LEAP2 Impairs the Capability of the Growth Hormone Secretagogue Receptor to Regulate the Dopamine 2 Receptor Signaling
Emilio Román Mustafá, Santiago Cordisco González, Marjorie Damian, Sonia Cantel, Séverine Denoyelle, Renaud Wagner, Helgi B. Schiöth, Jean‐Alain Fehrentz, Jean‐Louis Banères, Mario Perelló, Jesica Raingo
Abstract
The growth hormone secretagogue receptor (GHSR) signals in response to ghrelin, but also acts via ligand-independent mechanisms that include either constitutive activation or interaction with other G protein-coupled receptors, such as the dopamine 2 receptor (D2R). A key target of GHSR in neurons is voltage-gated calcium channels type 2.2 (Ca V 2.2). Recently, the liver-expressed antimicrobial peptide 2 (LEAP2) was recognized as a novel GHSR ligand, but the mechanism of action of LEAP2 on GHSR is not well understood. Here, we investigated the role of LEAP2 on the canonical and non-canonical modes of action of GHSR on Ca V 2.2 function. Using a heterologous expression system and patch-clamp recordings, we found that LEAP2 impairs the reduction of Ca V 2.2 currents induced by ghrelin-evoked and constitutive GHSR activities, acting as a GHSR antagonist and inverse agonist, respectively. We also found that LEAP2 prevents GHSR from modulating the effects of D2R signaling on Ca V 2.2 currents, and that the GHSR-binding N-terminal region LEAP2 underlies these effects. Using purified labeled receptors assembled into lipid nanodiscs and Forster Resonance Energy Transfer (FRET) assessments, we found that the N-terminal region of LEAP2 stabilizes an inactive conformation of GHSR that is dissociated from Gq protein and, consequently, reverses the effect of GHSR on D2R-dependent Gi activation. Thus, our results provide critical molecular insights into the mechanism mediating LEAP2 modulation of GHSR.