Background & Aims

Chronic pruritus is a major unmet clinical problem with one in four adults experiencing chronic pruritus in their lifetime1. The Gastrin releasing peptide receptor (GRPR), is a G protein-coupled receptor (GPCR) expressed in the spinal cord that regulates itch sensation. While GPCRs, like GRPR, are a major therapeutic target for itch relief2, there is a dearth of information about the mechanisms that regulate GRPR activation and signaling. The endosomal signaling of GPCRs provides new challenges for understanding how GPCR signaling is regulated, how endosomal signaling of GPCRs contributes to disease states like chronic pruritus and opens new targets for therapeutic development3. Here, we characterize the trafficking, endosomal recruitment of G proteins and subcellular signaling pathways of GRPR. Additionally, we demonstrate the importance of endosomal signaling of GRPR in animal models of itch and pruritus and use nanoparticles to specifically target endosomal signaling of GRPR.

Methods

The endosomal inhibitors (PitStop2 & Dyngo4a) and dominant negative forms of dynamin were used to inhibit endocytosis and trafficking of GRPR in HEK293 cells and in mouse models for itch. GRPR internalization and trafficking to endosomes was quantified using Bioluminescence resonance energy transfer (BRET) and fluorescent microscopy. Endosomal recruitment of G proteins and compartmentalized ERK signaling were quantified in cell lines using nanoBit BRET (nbBRET) and Forester resonance energy transfer assays (FRET). pH sensitive mesoporous silica nanoparticles (MSN) were used to deliver RC-3095, a GRPR antagonist, specifically to endosomes to block endosomal signaling of GRPR. Intrathecal injections of endosomal inhibitors, RC3095, empty MSN or MSN loaded with RC3095 (MSN-RC3095) were followed by intradermal injections of chloroquine (CQ). Scratching responses and normal motor responses were quantified using a mouse behavioral spectrometer.

Results

In HEK cells, activation of GRPR led to the rapid internalization and colocalization of GRPR with the early endosomal marker Rab5a. nbBRET assays show the GRP induced recruitment of the G-protein, G?q, but not G?s to the early endosomes. Pretreatment with endocytic inhibitors attenuated GRPR trafficking to endosomes, inhibited the GRP induced recruitment of G-protein to endosomes, and inhibited cytosolic and nuclear ERK signaling. Pre-incubation with MSN-3095 nanoparticles attenuated endosomal G-protein recruitment and blocked both cytosolic and nuclear ERK signaling. In mice, intradermal injections of CQ increased scratching bouts which were blocked by intrathecal injection of endosomal inhibitors Piststop2 and Dyngo4a in both male and female mice. Intrathecal injection of empty MSN or vehicle had no impact on CQ induced scratching. MSN-3095 injected 2 h before CQ completely abolished the scratching response but did not impact locomotor or grooming behaviors.

Conclusions

GRPR internalizes to the endosomal compartment following activation by GRP. GRPR is able to recruit G proteins to the endosome where GRPR induces cytosolic and nuclear ERK signaling. These results demonstrate a critical role for GRPR endosomal signaling in modulating cellular responses. In animal models, endocytic inhibitors blocked GRPR trafficking and inhibited CQ evoked scratching responses, demonstrating the importance of endosomal signaling of GRPR in the spinal cord and establish endosomal GRPR as a therapeutic target for pruritis. Additionally, pH sensitive nanoparticles with endosomal targeted release of GRPR antagonists attenuated cytosolic and nuclear ERK signaling, and completely blocked CQ induced scratching behavior. These results highlight the effectiveness of pH sensitive nanoparticles, like the MSN, to target and deliver antagonists to block GPCR signaling in endosomes and the need to target endosomal GPCR signaling in the development of therapeutics for chronic itch.

References

1Matterne, U., Apfelbacher, C. J., Vogelgsang, L., Loerbroks, A. & Weisshaar, E. Incidence and determinants of chronic pruritus: a population-based cohort study. Acta Derm Venereol 93, 532-537 (2013). https://doi.org/10.2340/00015555-1572
2Yosipovitch, G., Rosen, J. D. & Hashimoto, T. Itch: From mechanism to (novel) therapeutic approaches. J Allergy Clin Immunol 142, 1375-1390 (2018). https://doi.org/10.1016/j.jaci.2018.09.005
3Thomsen, A. R. B., Jensen, D. D., Hicks, G. A. & Bunnett, N. W. Therapeutic Targeting of Endosomal G-Protein-Coupled Receptors. Trends Pharmacol Sci 39, 879-891 (2018). https://doi.org/10.1016/j.tips.2018.08.003

Presenting Author

Dane D Jensen

Poster Authors

Dane Jensen

PhD

NYU College of Dentistry

Lead Author

E-mail

Jeffrey Retamal Santibañez

Laboratorio de Neurobiología, Facultad de Química y Biología, Universidad de Santiago de Chile

Lead Author

E-mail

Shavonne Teng

NYU

Lead Author

E-mail

Kam Leong

Department of Biomedical Engineering, Columbia University

Lead Author

E-mail

Topics

  • Specific Pain Conditions/Pain in Specific Populations: Itch