Orphan Receptor Gpr149 Modulates Activation of Primary Afferent Nociceptors

Background & Aims

Safe and effective therapies are needed to treat pain in any form – especially chronic pain states. This shortfall reflects, in part, our limited understanding of the interaction between known integrators of pain transduction (e.g. TRPV1) and a multitude of regulatory proteins and receptors in specialized sensory neurons that detect impending or actual tissue injury (primary afferent nociceptors). Moreover, despite their widespread use in society, the mechanism(s) by which the minor cannabinoid CBD acts on sensory neurons remain poorly understood. The orphan receptor Gpr149 gained our attention through its reported high level of expression in embryonic chick sensory ganglion [1] and its apparent co-expression with TRPV1, based on mining of a publicly available single-cell dorsal root ganglion (DRG) neuron data base [2]. Aim: We will test the hypothesis that orphan receptor Gpr149 is a minor cannabinoid – sensitive receptor that modulates nociceptor activation.

Methods

qRT-PCR was performed on single stranded cDNA derived from mouse DRG RNA (C57Bl/6) following established techniques [3, 4]. Measurement of Gpr149-directed Beta-arrestin 2 recruitment (activity) was accomplished by “Tango” methodology linking transcriptional output of luciferase gene expression to changes in Beta-arrestin 2 recruitment to the plasma membrane [5]. Minor cannabinoids: cannabidiol (CBD), cannabigerol (CBG), (Cayman Chemicals) were validated for purity and stability by HPLC. Mice (C57Bl/6, NaV1.8-Cre) were obtained from the Jackson Laboratory (Bar Harbor, ME) and Gpr149 lox/lox mice from Cyagen / Taconics (Germantown, New York) were crossed and housed within the UCSF animal care facility in a climate-controlled room on a 12 hr light/dark cycle. Lumbar DRGs harvested from adult male and female mice (C57Bl/6, Wt, Gpr149 Lox+/Lox- / NaV1.8) were cultured following established techniques and studied by microscopic calcium imaging [4].

Results

1) Based on data mining [2], we identified Gpr149 in a subset of peptide positive, DRG neurons that co-express TRPV1 and the cannabinoid receptor CB1R; 2) We determined by qRT-PCR that Gpr149 is selectively over-expressed in mouse DRG relative to brain, muscle, and kidney; 3) Using the “Tango” – based transcriptional luciferase assay, we observed Gpr149-directed constitutive Beta-arrestin 2 activity that was decreased by minor cannabinoids CBD and CBG; 4) CBD induced an increase in calcium responses in primary DRG neurons derived from Gpr149 heterozygous conditional knockdown mice (Gpr149 Lox+/- / Cre NaV1.8); 4) Primary DRG neurons derived from conditional knockdown mice (Gpr149 Lox+/- / Cre NaV1.8), were associated with decreased repetitive capsaicin-evoked (TRPV1) responses; 5) qRT-PCR analysis of DRG RNA derived from a homozygous strain of Gpr149 Lox+/+ / Cre NaV1.8 +/- mice demonstrated a near complete loss of Gpr149 mRNA expression.

Conclusions

We have advanced the hypothesis that Gpr149 is expressed as a nociceptor-specific receptor that modulates peripheral pain transduction. This was supported by transcriptomic analysis, quantitative measures of Gpr149 mRNA in sensory ganglion and its loss of expression following Nav1.8 – directed knockdown. Moreover, its role in nociception was supported by our observation that its genetic reduction in sensory neurons resulted in a decrease in responses following repetitive capsaicin applications. Our observations that minor cannabinoids decrease Gpr149 dependent Beta-arrestin 2 activity whereas knockdown of Gpr149 in Nav1.8 expressing nociceptors resulted in an increase in CBD- evoked calcium responses. This suggests a link between Gpr149, Beta-arrestin 2 and minor cannabinoid signaling. If genetic knockdown and/or pharmacologic modulation of Gpr149 can result in a decrease in nociceptor activation in vivo, Gpr149 may serve as a valuable target for future therapeutic development.

References

1.Friedel, R.H., et al., A novel 7-transmembrane receptor expressed in nerve growth factor-dependent sensory neurons. Mol Cell Neurosci, 2001. 17(1): p. 31-40.
2.Usoskin, D., et al., Unbiased classification of sensory neuron types by large-scale single-cell RNA sequencing. Nat Neurosci, 2015. 18(1): p. 145-53.
3.Sheehan, K., et al., Transcription factor Sp4 is required for hyperalgesic state persistence. PLoS One, 2019. 14(2): p. e0211349.
4.Xu, Z., et al., Chemotherapy for pain: reversing inflammatory and neuropathic pain with the anticancer agent mithramycin A. Pain, 2024. 165(1): p. 54-74.
5.Kroeze, W.K., et al., PRESTO-Tango as an open-source resource for interrogation of the druggable human GPCRome. Nat Struct Mol Biol, 2015. 22(5): p. 362-9.

Funding: R01 AT010757; HEAL Supplement AT010757- 02S1; California Bureau of Cannabis Control; UCSF Dept of Anesthesia and Perioperative Care.

Presenting Author