Regulatory importance of GHSR1a ghrelin receptor-dependent phosphorylation.

M Bouzo-Lorenzo1,2, BO Alen2, Y Pazos-Randulfe2, AB Tobin1, AJ Butcher1, JP Camiña2. 1MCR Toxicology Unit, Leicester, UK, 2IDICHUS, Santiago de Compostela, Spain

Ghrelin, a gastric peptide involved in growth hormone release and energy homeostasis, is the endogenous ligand of the G-protein coupled receptor (GPCR), growth hormone secretagogue receptor type 1a (GHS-R1a) which is mainly expressed in the pituitary and hypothalamus. Many studies have shown that ghrelin by its binding to GHSR1a, plays an important role for fundamental physiological functions including regulation of appetite, energy expenditure, fat accumulation, and growth hormone secretion. In addition GHS-R1a has received attention for its unusually high constitutive activity and its contribution to downstream signaling and physiological processes (1, 2).

Agonist regulated phosphorylation of GPCRs plays an essential role in the regulation of receptor function by promoting interactions of the receptor with β-arrestins and the subsequent activation of the intracellular signaling systems. The aim of this work was investigate wheter the GHSR1a becomes phosphorylated upon stimulation by ghrelin. This has been achieved using 32-P orthophosphate metabolic labelling and autoradiography. We found that addition of ghrelin (100nM) increased wild type GHSR1a phosphorylation 2.1 fold compared to basal levels (n=6). In addition, the ability of various synthetic ligands, truncations 1-5,1-14 and 1-18 of the ghrelin peptide, to induce phosphorylation of the receptor have also been tested. We found that these synthetic ligands increased receptor phosphorylation by 1.1,1.5 and 1.8 fold compared to basal levels respectively showing that increasing the number of amino acids in the ghrelin peptide increases thte ability of this peptide to induce receptor phosphorylation. Having determined that GHSR1a is a protein which becomes phosporylated upon agonist stimulation, the precise sites of phosphorylation induced by ghrelin stimulation were determined by generating tryptic phosphopeptides and analysing these peptides by liquid chromotography coupled to tandem mass spectrometry (LC-ms/ms). LC-ms/ms analysis identified 5 sites of phosphorylation all of which were located in the receptor C-terminal tail (S349,T350,S362,S363 and T366).These sites form 2 “clusters” of phosphorylation in the C-tail of GHSR1a. To investigate the role of phosphorylation in fuction of the GHSR1a receptor we have generated serine to alanine or threonine to alanine mutations of all these sites thus generating a mutated GHSR1a (GHSR1a S to A) which can no longer become phosphorylated upon ghrelin stimulation. Using GHSR1a constructs tagged to green fluorescent protein (EGFP) and expressed in HEK293 cells, we show that the GHSR1a S to A traffics more efficiently to the plasma membrane, shows no constitutive internalization compared to WT GHSR1a and shows no agonist regulated receptor internalization compares to the WT GHSR1a which is completely internalized after 20 minutes of stimulation with ghrelin.

This work provides an insight into the importance of agonist regulated receptor phosphorylation in regulating GPCR function. Moreover, further characterization of the signaling system associated with β-arrestin and receptor internalization could lead us to a correct design of ligands associated with the functionality of these receptors.

1. Holst B et al., Mol Endocrinol. 17(11):2201-10, 2003

2. Mear Y et al., Front Neurosci. 29;7:87, 2013