Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Jun;32(6):3184-3192.
doi: 10.1096/fj.201700337RR. Epub 2018 Jan 22.

Receptor-specific crosstalk between prostanoid E receptor 3 and bombesin receptor subtype 3

Yan Zhang  1 , Yanfang Liu  2 , Lehao Wu  1 , Chao Fan  1 , Zhiwei Wang  3 , Xiuli Zhang  2 , Amal Alachkar  3 , Xinmiao Liang  2 , Olivier Civelli  3
Affiliations

Receptor-specific crosstalk between prostanoid E receptor 3 and bombesin receptor subtype 3

Yan Zhang et al. FASEB J. 2018 Jun.

Abstract

Bombesin receptor subtype 3 (BRS-3) is a GPCR that is expressed in the CNS, peripheral tissues, and tumors. Our understanding of BRS-3's role in physiology and pathophysiology is limited because its natural ligand is unknown. In an attempt to identify this ligand, we screened toad skin ( Bufo bufo gargarizans Cantor) extracts and identified prostaglandins as putative ligands. In BRS-3-transfected human embryonic kidney (HEK) cells, we found that prostaglandins, with prostaglandin E2 (PGE2) being the most potent, fulfill the pharmacologic criteria of affinity, selectivity, and specificity to be considered as agonists to the BRS-3 receptor. However, PGE2 is unable to activate BRS-3 in different cellular environments. We speculated that EP receptors might be the cause of this cellular selectivity, and we found that EP3 is the receptor primarily responsible for the differential PGE2 effect. Consequently, we reconstituted the HEK environment in Chinese hamster ovary (CHO) cells and found that BRS-3 and EP3 interact to potentiate PGE2 signaling. This potentiating effect is receptor specific, and it occurs only when BRS-3 is paired to EP3. Our study represents an example of functional crosstalk between two distantly related GPCRs and may be of clinical importance for BRS-3-targeted therapies.-Zhang, Y., Liu, Y., Wu, L., Fan, C., Wang, Z., Zhang, X., Alachkar, A., Liang, X., Civelli, O. Receptor-specific crosstalk between prostanoid E receptor 3 and bombesin receptor subtype 3.

Keywords: BRS-3 receptor; EP3 receptor; PGE2; toad skin.

PubMed Disclaimer

Conflict of interest statement

This work was supported by U.S. National Institutes of Health (NIH) National Institute on Drug Abuse Grant DA024746, and the Eric L. and Lila D. Nelson Chair in Neuropharmacology (to O.C.). This work was also supported by the National Natural Science Foundation of China Key Program 21135005 (to X.L.), and Grant 81402806 (to Y.Z.). The authors declare no conflicts of interest.

Figures

Figure 1.
Figure 1.
Isolation and characterization of potential BRS-3 ligand from toad skin. A) Elution profile of toad skin extract on C18HCE reverse-phase HPLC column (4.6 × 150 mm, 5 μm) and activities of 48 fractions (1 min) tested for their abilities to induce intracellular Ca2+ mobilization in HEK293 cells expressing BRS-3 receptor. Signal is expressed as ratio of increment obtained by BRS-3–transfected HEK293 cells vs. HEK293 wild-type cells. Elution was performed with linear gradient from 5 to 15% CH3CN in 30 min, then from 15T to 95% CH3CN in 10 min. Flow rate was 1 ml/min. Fraction 46 (arrow) was most active. B) PGB2 purification and structure elucidation. Inset shows structure of PGB2 and its response in BRS-3/HEK monitored by Ca2+ mobilization. Error bars represent sem of triplicate measurements for each point.
Figure 2.
Figure 2.
Prostaglandin activation of BRS-3. A) Prostaglandins dose–response curve in HEK cells stably expressing BRS-3 receptors. BRS-3 agonist 16a was used as positive control. B) Inhibitory effects of BRS-3 antagonists on PGE2 induced Ca2+ mobilization in BRS-3 stable cells. Bantag-1 dose-dependently inhibits PGE2 (5 nM) induced response (left). NMB receptor antagonist D-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-NaI-NH2 dose-dependently inhibits PGE2 (5 nM) induced response (right). C) No PGE2 induced Ca2+ mobilization in HEK cells expressing NMB (left) and GRP receptors (right). Error bars represent sem of triplicate measurements for each point.
Figure 3.
Figure 3.
No effects of PGE2 may be observed in CHO-BRS-3 or HeLa-BRS-3 cells; 16a was used as positive control.
Figure 4.
Figure 4.
Crosstalk between BRS-3 and EP3 receptors in CHO cells. A) Enhanced response to PGE2 (1 μM) in CHO-EP3 (2 μg)/BRS-3 (5 μg) cells. B, C) Dose–response of BRS-3 (B) and EP3 (C) on PGE2 (1 μM) induced Ca2+ influx in CHO-EP3/BRS-3. D, E) Effects of EP3 (L-798,106, 10 μM; Sigma-Aldrich) and BRS-3 antagonists (bantag-1, 10 μM; Sigma-Aldrich) on PGE2 (1 μM) (D) or 16a (1 μM) (E) induced Ca2+ influx in CHO-EP3 (2 μg)/BRS-3 (5 μg) cells. F) No inhibitory effect of bantag-1 on PGE2 response in CHO-EP3 cells. Error bars represent sem of triplicate measurements for each point.
Figure 5.
Figure 5.
Specificity of EP3/BRS-3 receptor pair and G protein involved. No enhancement of PGE2 induced Ca2+ mobilization in (A) CHO-EP1/hBRS-3 cells, (B) CHO-EP2/hBRS-3 cells, (C) CHO-EP3/MCH, and (D) CHO-EP3/NMB cells. E) Effect of PTX (100 ng/ml) treatment on PGE2 induced Ca2+ mobilization in CHO-EP3/BRS-3. Error bars represent sem of triplicate measurements for each point.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Fathi Z., Corjay M. H., Shapira H., Wada E., Benya R., Jensen R., Viallet J., Sausville E. A., Battey J. F. (1993) BRS-3: a novel bombesin receptor subtype selectively expressed in testis and lung carcinoma cells. J. Biol. Chem. 268, 5979–5984 - PubMed
    1. Sano H., Feighner S. D., Hreniuk D. L., Iwaasa H., Sailer A. W., Pan J., Reitman M. L., Kanatani A., Howard A. D., Tan C. P. (2004) Characterization of the bombesin-like peptide receptor family in primates. Genomics 84, 139–146 10.1016/j.ygeno.2004.01.008 - DOI - PubMed
    1. Porcher C., Juhem A., Peinnequin A., Bonaz B. (2005) Bombesin receptor subtype-3 is expressed by the enteric nervous system and by interstitial cells of Cajal in the rat gastrointestinal tract. Cell Tissue Res. 320, 21–31 10.1007/s00441-004-1032-1 - DOI - PubMed
    1. Ohki-Hamazaki H., Watase K., Yamamoto K., Ogura H., Yamano M., Yamada K., Maeno H., Imaki J., Kikuyama S., Wada E., Wada K. (1997) Mice lacking bombesin receptor subtype-3 develop metabolic defects and obesity. Nature 390, 165–169 10.1038/36568 - DOI - PubMed
    1. Jensen R. T., Battey J. F., Spindel E. R., Benya R. V. (2008) International Union of Pharmacology. LXVIII. Mammalian bombesin receptors: nomenclature, distribution, pharmacology, signaling, and functions in normal and disease states. Pharmacol. Rev. 60, 1–42 10.1124/pr.107.07108 - DOI - PMC - PubMed

Publication types

Substances