Introduction

Ghrelin, a 28-amino acid gut hormone, occurs in acylated (AG) and unacylated (UAG) variants. AG, acting via the growth hormone secretagogue receptor (GHSR1a), induces food intake and is diabetogenic. UAG counteracts these effects through unknown mechanisms. While screening for potential UAG receptor(s) we discovered previously uncharacterised interactions of AG and UAG with five cell membrane proteins (MPs), three of which are known to modulate metabolism. Here, we have studied if these MPs affect AG signalling.

Methods

To investigate modulation of GHSR1a signalling by the MPs in response to AG we transfected HEK293 cells with GHSR1a ± MP expression plasmids. As GHSR1a is coupled with calcium signalling via Gq proteins, aequorin luminescence was used to evaluate Ca²⁺ influx into the cells. To investigate potential interactions between the receptor and the MPs we expressed HA- and FLAG-tagged proteins in HEK293 cells. Cell lysates were then used for co-immunoprecipitation studies using either anti-HA or anti-FLAG antibody conjugated beads. Bead eluents were analysed by Western blot.

Results

Two of the MPs markedly enhanced the efficacy (2.5-to-5.5-fold), but not the potency, of AG-induced Ca²⁺ influx. The magnitude of the effect related to the amount of transfected MP expression plasmid, and is dependent on the presence of GHSR1a. UAG treatment did not alter the effect. Ectodomain shedding, an important feature of these MPs, dose-dependently reduced Ca²⁺ influx in MP-GHSR1a transfected cells. Preliminary co-immunoprecipitation studies suggest an interaction between the intracellular domain of one of the MPs and GHSR1a.

Conclusions

Two of the newly identified MPs markedly enhance the response of the ghrelin receptor to AG stimulation, indicating that these MPs are either co-receptors or intracellular modulators of downstream GHSR1a signalling. The mechanism by which these proteins influence signalling remains to be fully elucidated, but may involve an intra-cellular interaction.