Technical Comments

Comment on "Obestatin, a Peptide Encoded by the Ghrelin Gene, Opposes Ghrelin's Effects on Food Intake"

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Science  09 Feb 2007:
Vol. 315, Issue 5813, pp. 766
DOI: 10.1126/science.1135047

Abstract

Zhang et al. (Research Articles, 11 November 2005, p. 996) reported that obestatin, a peptide derived from the ghrelin precursor, activated the orphan G protein–coupled receptor GPR39. However, we found that I125-obestatin does not bind GPR39 and observed no effects of obestatin on GPR39-transfected cells in various functional assays (cyclic adenosine monophosphate production, calcium mobilization, and GPR39 internalization). Our results indicate that obestatin is not the cognate ligand for GPR39.

Zhang et al. (1) reported the identification, using a bioinformatic approach, of a novel neuropeptide designated obestatin, which is derived from the ghrelin precursor (1). They subsequently purified obestatin from a rat stomach extract and reported that obestatin is a cognate ligand for the orphan G protein–coupled receptor (GPCR) GPR39. Here, we provide independent evidence that obestatin does not interact with GPR39.

To investigate whether obestatin is the natural ligand of GPR39, we transfected Chinese hamster ovary (CHO) cells with human GPR39 cDNA [accession no. AAC26082, corresponding to the GPR39 sequence reported in (1) (fig. S1)]. We established two stably transfected CHO cell lines expressing either the Flag-tagged or the enhanced green fluorescent protein (EGFP)–tagged human GPR39. The integration of human GPR39 cDNA in the genome of the two cell lines and the presence of the GPR39 mRNA were confirmed (fig. S2 A and B), and the correct expression of the receptor at the cell surface was visualized by confocal microscopy (fig. S3 A and D). We subsequently synthesized human obestatin and thoroughly characterized the synthetic peptide by mass spectrometry and microsequencing. Its effects were then tested in parallel with those of a commercial source of synthetic human obestatin (Phoenix Pharmaceuticals, Inc., Belmont, California).

Zhang et al. (1) showed that I125-obestatin binds with high affinity to crude plasma-membrane preparations from rat jejunum, stomach, ileum, hypothalamus, pituitary, and CHO cells transfected with human GPR39. We tested the ability of commercially available I125-obestatin (Phoenix Pharmaceuticals, Inc.) to interact with GPR39 in our two GPR39-CHO cell lines. In our hands, no specific binding was observed with I125-obestatin at a concentration of 5 × 10–10 M, which is half the dissociation constant reported in (1) (Fig. 1). These experiments were conducted under similar incubation conditions as those used by Zhang et al. (1). We also found no evidence of specific binding in crude pituitary membranes (Fig. 1A).

Fig. 1.

Binding of obestatin on GPR39-transfected CHO cell membranes and effects of obestatin on cAMP production in CHO cells stably or transiently expressing GPR39. (A) Membranes from transfected CHO cells or from pituitary cells were tested for their ability to bind I125-obestatin using the protocol described by Zhang et al. (1): (1) naive CHO cells, (2) flag-tagged GPR39-expressing CHO cells, (3) EGFP-tagged GPR39-expressing CHO cells, and (4) pituitary cell membranes. Experiments were performed in triplicate and reproduced independently twice. Black bars, total binding; open bars, 10–6 M obestatin. (B to D) CHO cells stably expressing either the EGFP-tagged (B) or the Flag-tagged (C) GPR39, or CHO cells transiently transfected with the human untagged GPR39 (D) were incubated for 1 or 16 hours in the absence or presence of 10–6 or 10–5 M obestatin before cAMP production assessment.

Zhang et al. (1) reported the activation of cAMP production in CHO and human embryonic kidney (HEK) 293 cells transiently transfected with human GPR39 after a 16-hour incubation with human obestatin, with maximal stimulation at obestatin concentrations between 3 × 10–8 and 10–7 M. Either using the same experimental conditions or using a similar protocol with an incubation time for obestatin reduced to 1 hour, we found that obestatin (10–5, 10–6 M) did not induce a significant increase in cAMP formation in CHO cells stably expressing the Flag- or EGFP-tagged GPR39 or in CHO cells transientlytransfected with the untagged GPR39 (Fig. 1, B to D). However, treatment of the cells with 10–5 M forskolin provoked a robust stimulation of cAMP production (Fig. 1D). In addition, incubation of CHO cells stably expressing the Flag- or EGFP-tagged GPR39 with human obestatin for 20 to 60 min, at doses ranging from 10–9 to 10–6 M, did not modify intracellular calcium concentration (fig. S3, C and F) and did not promote GPR39 internalization (fig. S3, B and E).

In conclusion, although Zhang et al. (1) provided convincing evidence for processing of the ghrelin precursor to generate obestatin in vivo, the lack of activity of the synthetic peptide in various GPR39 functional assays suggests that obestatin is not the endogenous ligand of the orphan receptor GPR39. Similar observations have now been reported by other laboratories (2, 3).

Supporting Online Material

www.sciencemag.org/cgi/content/full/315/5813/766c/DC1

Materials and Methods

Figs. S1 to S3

References and Notes

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