The GPCR accessory protein MRAP2 regulates both biased signaling and constitutive activity of the ghrelin receptor GHSR1a.

Ghrelin is a hormone secreted by the stomach during fasting periods and acts through its receptor, the growth hormone secretagogue 1a (GHSR1a), to promote food intake and prevent hypoglycemia. As such, GHSR1a is an important regulator of energy and glucose homeostasis and a target for the treatment of obesity. Here, we showed that the accessory protein MRAP2 altered GHSR1a signaling by inhibiting its constitutive activity, as well as by enhancing its G protein-dependent signaling and blocking the recruitment and signaling of ß-arrestin in response to ghrelin. In addition, the effects of MRAP2 on the Gαq and ß-arrestin pathways were independent and involved distinct regions of MRAP2. These findings may have implications for the regulation of ghrelin function in vivo and the role of MRAP2 in energy homeostasis. They also show that accessory proteins can bias signaling downstream of GPCRs in response to their endogenous agonist.

Ghrelin Receptor Influence on Cocaine Reward is Not Directly Dependent on Peripheral Acyl-Ghrelin.

The peptide hormone acyl-ghrelin and its receptor, GHSR1a, represent intriguing therapeutic targets due to their actions in metabolic homeostasis and reward activity. However, this pleotropic activity makes it difficult to intervene in this system without inducing unwanted effects. Thus, it is desirable to identify passive and active regulatory mechanisms that allow differentiation between functional domains. Anatomical restriction by the blood brain barrier represents one major passive regulatory mechanism. However, it is likely that the ghrelin system is subject to additional passive mechanisms that promote independent regulation of orexigenic behavior and reward processing. By applying acyl-ghrelin sequestering antibodies, it was determined that peripheral sequestration of acyl-ghrelin is sufficient to blunt weight gain, but not cocaine rewarding effects. However, both weight gain and reward-associated behaviors were shown to be blocked by direct antagonism of GHSR1a. Overall, these data indicate that GHSR1a effects on reward are independent from peripheral acyl-ghrelin binding, whereas centrally-mediated alteration of energy storage requires peripheral acyl-ghrelin binding. This demonstration of variable ligand-dependence amongst functionally-distinct GHSR1a populations is used to generate a regulatory model for functional manipulation of specific effects when attempting to therapeutically target the ghrelin system.

The Impact of Ghrelin in Metabolic Diseases: An Immune Perspective.

Obesity and insulin resistance have reached epidemic proportions. Obesogenic conditions are associated with increased risk for the development of other comorbidities and obesity-related diseases. In metabolic disorders, there is chronic low-grade inflammation induced by the activation of immune cells, especially in metabolic relevant organs such as white adipose tissue (WAT). These immune cells are regulated by environmental and systemic cues. Ghrelin is a peptide secreted mainly by X/A-like gastric cells and acts through the growth hormone secretagogue receptor (GHS-R). This receptor is broadly expressed in the central nervous system (CNS) and in several cell types, including immune cells. Studies show that ghrelin induces an orexigenic state, and there is increasing evidence implicating an immunoregulatory role for ghrelin. Ghrelin mainly acts on the innate and adaptive immune systems to suppress inflammation and induce an anti-inflammatory profile. In this review, we discuss the immunoregulatory roles of ghrelin, the mechanisms by which ghrelin acts and potential pharmacological applications for ghrelin in the treatment of obesity-associated inflammatory diseases, such as type 2 diabetes (T2D).

Ghrelin receptor modulates T helper cells during intestinal inflammation.

BACKGROUND: The orexigenic peptide ghrelin has anti-inflammatory properties in colitis, however, the mechanism of action and the immune cells targeted remain still to be elucidated. Here, we assessed the possible effect of ghrelin on T helper (Th) cells in a T cell transfer model of chronic colitis. METHODS: Disease was induced in the recombination activating gene 1 knockout mice (Rag1(-/-) ) by adoptive transfer of naïve Th cells from ghrelin receptor knockout mice (GRLN-R(-/-) ) or littermate wild-type (WT) mice. The course and severity of colitis was assessed by monitoring body weight, diarrhea score, histological analysis, gene expression, and flow cytometry analysis. The possible effects of ghrelin on Th cell proliferation, polarization, and apoptosis was examined in vitro. KEY RESULTS: Our data showed that Rag1(-/-) mice injected with GRLN-R(-/-) Th cells displayed increased severity of colitis compared to mice injected with WT Th cells. In addition, Rag1(-/-) mice injected with GRLN-R(-/-) Th cells had significantly higher intestinal inflammation and increased accumulation of Th1 and Th17 cells in the colon. In vitro, ghrelin directly affected proliferation of Th cells and induced apoptosis whereas it did not influence Th cell polarization. CONCLUSION & INFERENCES: Our observations suggest that ghrelin modulates Th effector cells in the gut controlling proliferation and inducing apoptosis. Our findings further support the use of ghrelin as a novel therapeutic option to treat intestinal inflammatory diseases.

Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay.

G-protein-coupled receptors (GPCRs) are one of the most important drug targets, and anti-GPCR monoclonal antibody (mAb) is an essential tool for functional analysis of GPCRs. However, it is very difficult to develop GPCR-specific mAbs due to difficulties in production of recombinant GPCR antigens, and lack of efficient mAb screening method. Here we describe a novel approach for the production of mAbs against GPCR using two original methods, bilayer-dialysis method and biotinylated liposome-based interaction assay (BiLIA), both of which are developed using wheat cell-free protein synthesis system and liposome technology. Using bilayer-dialysis method, various GPCRs were successfully synthesized with quality and quantity sufficient for immunization. For selection of specific mAb, we designed BiLIA that detects interaction between antibody and membrane protein on liposome. BiLIA prevented denaturation of GPCR, and then preferably selected conformation-sensitive antibodies. Using this approach, we successfully obtained mAbs against DRD1, GHSR, PTGER1 and T1R1. With respect to DRD1 mAb, 36 mouse mAbs and 6 rabbit mAbs were obtained which specifically recognized native DRD1 with high affinity. Among them, half of the mAbs were conformation-sensitive mAb, and two mAbs recognized extracellular loop 2 of DRD1. These results indicated that this approach is useful for GPCR mAb production.

Ghrelin and the gut.

Ghrelin is the endogenous ligand for the growth hormone secretagogue receptor (GHS-R) which was identified in the pituitary gland and is now named the ghrelin receptor. However, the peptide is most abundant in the stomach and ghrelin receptors are present in all major organ systems and tissues. Ghrelin forms with motilin, a new gut peptide family and the sequence similarities of peptides and receptors suggest they evolved by gene duplication. Nevertheless, no cross-reactivity exits between both peptides. Ghrelin shares with motilin motor effects in the gut, in particular gastric emptying and the induction of the migrating motor complex, but ghrelin also affects gastric acid secretion, offers gastroprotection and may modulate intestinal inflammation. The effects of ghrelin result from the activation of central, vagal and enteric neural receptors and receptors on immune cells. Ghrelin agonists have been developed for the treatment of hypomotility disorders and the peptidomimetic TZP-102 is in phase 2 clinical trials for diabetic gastroparesis.

Association study of ghrelin receptor gene polymorphisms in rheumatoid arthritis.

OBJECTIVES: Ghrelin is a newly characterised growth hormone (GH) releasing peptide widely distributed that may play an important role in the regulation of metabolic balance in inflammatory diseases such as rheumatoid arthritis (RA) by decreasing the pro-inflammatory Th1 responses. In this study we investigated the possible contribution of several polymorphisms in the functional Ghrelin receptor to RA susceptibility. METHODS: A screening of 3 single nucleotide polymorphisms (SNPs) was performed in a total of 950 RA patients and 990 healthy controls of Spanish Caucasian origin. Genotyping of all 3 SNPs was performed by real-time polymerase chain reaction technology, using the TaqMan 5'-allele discrimination assay. RESULTS: We observed no statistically significant deviation between RA patients and controls for the GHSR SNPs analysed. In addition, we performed a haplotype analysis that did not reveal an association with RA susceptibility. The stratification analysis for the presence of shared epitope (SE), rheumatoid factor (RF) or antibodies anti cyclic citrullinated peptide (anti-CCP) did not detect significant association of the GHSR polymorphisms with RA. CONCLUSIONS: These findings suggest that the GHSR gene polymorphisms do not appear to play a major role in RA genetic predisposition in our population.

Novel connections between the neuroendocrine and immune systems: the ghrelin immunoregulatory network.

There appears to be bidirectional communication between the neuroendocrine and immune systems. This communication is mediated by way of an array of cytokines, hormones, and neuropeptides. Inflammatory cytokines released by immune cells have been shown to act on the central nervous system to control food intake and energy homeostasis. Decrease in food intake or anorexia is one of the most common symptoms of illness, injury, or inflammation. The adipocyte-derived hormone, leptin, is considered a critical sensory anorexigenic mediator that signals to the brain changes in stored energy, determined by an altered balance between food intake and energy expenditure and has been shown to exert certain proinflammatory effects on immune cells. In contrast, ghrelin, the endogenous ligand for growth hormone secretagogue receptors (GHSRs), is produced primarily from stomach serving as a potent circulating orexigen controlling energy expenditure, adiposity, and GH secretion. However, the functional role of ghrelin and GHS in immune cell function remains unclear. Here, we review the current literature supporting a role for ghrelin in controlling inflammation and immunity and the potential therapeutic use of ghrelin and GHSR agonists in the management of inflammation and in restoration of thymic function in immunocompromised individuals.