Common Structural Basis for Constitutive Activity of the Ghrelin Receptor Family

Abstract

Three members of the ghrelin receptor family were characterized in parallel: the ghrelin receptor, the neurotensin receptor 2 and the orphan receptor GPR39. In transiently transfected COS-7 and human embryonic kidney 293 cells, all three receptors displayed a high degree of ligand-independent signaling activity. The structurally homologous motilin receptor served as a constitutively silent control; upon agonist stimulation, however, it signaled with a similar efficacy to the three related receptors. The constitutive activity of the ghrelin receptor and of neurotensin receptor 2 through the G q , phospholipase C pathway was ϳ50% of their maximal capacity as determined through inositol phosphate accumulation. These two receptors also showed very high constitutive activity in activation of cAMP response element-driven transcription. GPR39 displayed a clear but lower degree of constitutive activity through the inositol phosphate and cAMP response element pathways. In contrast, GPR39 signaled with the highest constitutive activity in respect of activation of serum response element-dependent transcription, in part, possibly, through G 12/13 and Rho kinase. Antibody feeding experiments demonstrated that the epitope-tagged ghrelin receptor was constitutively internalized but could be trapped at the cell surface by an inverse agonist, whereas GPR39 remained at the cell surface. Mutational analysis showed that the constitutive activity of both the ghrelin receptor and GPR39 could systematically be tuned up and down depending on the size and hydrophobicity of the side chain in position VI:16 in the context of an aromatic residue at VII:09 and a large hydrophobic residue at VII:06. It is concluded that the three ghrelin-like receptors display an unusually high degree of constitutive activity, the structural basis for which is determined by an aromatic cluster on the inner face of the extracellular ends of TMs VI and VII.

Interactions between neurotensin receptors and G proteins

“…In these cells like in CHO cells, NT increased Erk1/2 activation. However, the NT effect was of small amplitude compared with those obtained for other receptors of the same family, such as ghrelin receptors [30]. SR 48692 was not evaluated in this work.…”

“…By contrast with InsP production, human NTS2 expressed in COS cells was not constitutively active for Erk1/2 activation [30]. In these cells like in CHO cells, NT increased Erk1/2 activation.…”

“…SR 48692 retained agonist activity towards this transduction pathway, NT behaved as a neutral antagonist, and levocabastine as a weak partial inverse agonist [62]. When expressed in HEK-293 cells [30], human NTS2 constitutively activated serum response element (SRE) signaling through both pertussis-sensitive G proteins and Rho-GTPases, suggesting coupling to G i/o and G 12/13 -type G proteins. Here again, NT behaved as a neutral antagonist on the constitutive signal.…”

“…It can be suggested that human NTS2 may interact with G q/11 [30,62,76], G i/o [30] and G 12/13 [30]. Whether interaction with G i/o might account for arachidonic acid release [76], as observed with NTS1 [53], remains to be established.…”

“…The constitutive activity of human NTS2 towards InsP production [62] and SRE signaling [30] suggested a ligand-independent activation of G q/11 , G i/o and perhaps G 12/13 . It is not clear whether constitutive activity towards InsP production does not occur in CHO cells or was just missed in previous studies [76].…”