Abstract
The truncated non-signaling ghrelin receptor growth hormone secretagogue R1b (GHS-R1b) has been suggested to simply exert a dominant negative role in the trafficking and signaling of the full and functional ghrelin receptor GHS-R1a. Here we reveal a more complex modulatory role of GHS-R1b. Differential co-expression of GHS-R1a and GHS-R1b, both in HEK-293T cells and in striatal and hippocampal neurons in culture, demonstrates that GHS-R1b acts as a dual modulator of GHS-R1a function: low relative GHS-R1b expression potentiates and high relative GHS-R1b expression inhibits GHS-R1a function by facilitating GHS-R1a trafficking to the plasma membrane and by exerting a negative allosteric effect on GHS-R1a signaling, respectively. We found a preferential Gi/o coupling of the GHS-R1a-GHS-R1b complex in HEK-293T cells and, unexpectedly, a preferential Gs/olf coupling in both striatal and hippocampal neurons in culture. A dopamine D1 receptor (D1R) antagonist blocked ghrelin-induced cAMP accumulation in striatal but not hippocampal neurons, indicating the involvement of D1R in the striatal GHS-R1a-Gs/olf coupling. Experiments in HEK-293T cells demonstrated that D1R co-expression promotes a switch in GHS-R1a-G protein coupling from Gi/o to Gs/olf, but only upon co-expression of GHS-R1b. Furthermore, resonance energy transfer experiments showed that D1R interacts with GHS-R1a, but only in the presence of GHS-R1b. Therefore, GHS-R1b not only determines the efficacy of ghrelin-induced GHS-R1a-mediated signaling but also determines the ability of GHS-R1a to form oligomeric complexes with other receptors, promoting profound qualitative changes in ghrelin-induced signaling.
Highlights
The truncated non-signaling ghrelin receptor growth hormone secretagogue R1b (GHS-R1b) has been suggested to exert a dominant negative role in the trafficking and signaling of the full and functional ghrelin receptor GHS-R1a
Differential co-expression of GHS-R1a and GHS-R1b, both in HEK293T cells and in striatal and hippocampal neurons in culture, demonstrates that GHS-R1b acts as a dual modulator of GHSR1a function: low relative GHS-R1b expression potentiates and high relative GHS-R1b expression inhibits GHS-R1a function by facilitating GHS-R1a trafficking to the plasma membrane and by exerting a negative allosteric effect on GHS-R1a signaling, respectively
Taking into account the results of biotinylation experiments, these results suggest that low relative GHS-R1b expression potentiates ghrelin-induced Gi/o protein-mediated signaling by facilitating GHS-R1a trafficking to the plasma membrane
Summary
The truncated non-signaling ghrelin receptor growth hormone secretagogue R1b (GHS-R1b) has been suggested to exert a dominant negative role in the trafficking and signaling of the full and functional ghrelin receptor GHS-R1a. Two different mechanisms have been invoked for the dominant negative function of GHS-R1b: intracellular retention by an inability of GHS-R1b to traffic to the plasma membrane [6, 7] and an allosteric mechanism that produces a conformational block of GHS-R1a in a non-signaling conformation [5]. Those results were obtained from in vitro experiments in transfected cells and in reconstituted lipid vesicles, and, either mechanism could be involved in a physiological cellular environment. An unexpected additional finding in striatal and hippocampal neurons in culture was a predominant Gs/olf protein-dependent signaling of ghrelin that, in striatal neurons, depended on dopamine D1 receptor (D1R)-GHS-R1a-GHS-R1b heteromerization
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