A Characterization of the Underlying Regulatory Mechanism
of the CB1R – 5-HT2AR Heterodimerization Process
Rania Belhadjhamida, Mustafa Sherik, and Eesha Lodhi
Abstract
The activation of cannabinoid receptors 1 (CB1R) by delta-9-
tetrahydrocannabinol (THC) induces analgesia. While beneficial as a
therapeutic agent, simulation by THC also induces the negative
response of memory impairment. By separating the undesirable effect
from the beneficial property, THC-based remedies may prove
invaluable in the treatment of memory disorders and chronic pain.
Behavioural studies carried out in serotonin 2A receptor (5-HT2A) knock-
out mice revealed the effects of memory deficit are controlled by 5-
HT2A only when dimerization occurs with CB1R in the hippocampus and
prefrontal cortex. While previous literature has been able to depict the
effects of each receptor upon dimer formation, the mechanisms of
regulation and translocation of dimerization remains unknown. This is
based on the fact that the lack of dimerization between the receptors
in the rest of the brain is not simply the result of lack of expression,
since both receptors are present and functional, but an underlying
mechanism that regulates dimerization. By examining assessments of
regulatory mechanisms of similarly classified receptors in previous
literature, our goal is to devise our own method of characterizing the
regulatory induction pathway. This will be accomplished by conducting
a multi-point study using FRET and quantification experiments from the
starting point of receptor biosynthesis in the endoplasmic reticulum up
to incorporation into the plasma membrane and to interaction with
added THC agonist. The time at which dimerization occurs will provide
insight into the specific location from which further assessment of the
regulatory mechanism(s) can be conducted.
Background
§ Cannabinoid receptor 1 (CB1R) produces analgesics
§ Serotonin receptor (5-HT2A) involved in mood and anxiety
§ CB1R and 5-HT2A receptors are G protein-coupled receptors (GPCR)
• G proteins are guanine nucleotide-binding proteins
• G proteins transmit signals from outside to inside the cell
§ Receptors form heteromers are are expressed in hippocampus
• Hippocampus associated with memory
• Cerebral cortex associated with pain
Adapted from Vinals, 2015.
§ Heteromers required for amnesic effects, but NOT analgesics
• Complex formation interrupted by interfering peptide, gene-deletion
of 5-HT2A, and cross-antagonism using 5-HT2A antagonist
• Decrease in extracellular signal-regulated kinases (ERK) compared to
upregulation when receptors activated individually
§ Lack of dimerization in the rest of the brain not because lack of expression
• Present and functional in all parts
• Underlying regulatory mechanism exists
Experimental Design
Methodology
a) In vitro culture of hippocampal neurons from prenatal wild-type mice
[Seibenhenner, 2012] will be used to characterize the regulation of
hetero-dimerization between CB1 and 5-HT2A receptors.
I. ThermoFisher Scientific CRISPR-Cas9 Kit will be used to add YFP
sequence to the host 5HT2A receptor gene.
II. Genetic code expansion technology [Park, 2019] will be used to
incorporate ANAP, a fluorescent amino acid, into CB1 receptor
gene.
b) Dimerization will be assessed in 30 second intervals starting from
biosynthesis to integration into the membrane using FRET based on
fluorophores of the receptors.
• Initial excitation of ANAP at 405 nm
• ANAP fluorescence measured at 410-530 nm
• FRET measured at 550-620 nm
• Absence or presence of emission from the ANAP will
determine the dimerization status, as it will indicate the
transfer of energy between the receptors.
• Intensity of the FRET signal (emission) will indicate the
distance at which this interaction occurs.
c) At time point three, FRET will assess the addition of 2 micromolar
of THC, for activation of receptors, through electroporation [Anavi-
Goffer, 2011].
Hypothesis
We hypothesize that if FRET system and ERK quantification can be
applied to determine dimerization states, then the time point of
heterodimerization between the CB1 and 5-HT2A receptors’ synthesis and
incorporation into the cellular membrane will be determined. This will clarify
the location in the cell in which dimerization occurs and provide more insight
into the mode of regulation of dimerization.
Aims and Rationale
Aim: Identify the location of CB1R – 5-HT2A dimerization using a FRET
assessment at time points of biosynthesis, trafficking and integration into the
plasma membrane.
Rationale: Characterization of a time-point in which a hetero-dimerization
event occurs will detail the location of the event in the cell. This will provide
insight as to which stage of the biosynthesis, trafficking and THC activation
process of the receptors at which dimerization occurs. As such, this will detail
the factors that contribute to the regulatory mode of dimerization.
• Established literature shows that class-C GPCRs commonly dimerize
during biosynthesis while class-A GPCRs dimerize upon ligand binding
(Milligan, 2007; Bai, 2004).
• Establishment of this modulatory pathway may potentially reveal
alternative pathways for pharmacological synthesis of medicinal
marijuana-based drugs that provide solely analgesic effects.
§ Quantification of ERK signaling pathway ensures that the receptors have
made a stable heterodimer complex.
• Previous studies have established that attenuation of ERK signaling
pathway upon dimerization indicates change in function.
Conclusion
THC is a key ingredient in the psychoactive properties of marijuana
and in the pursuit to harness it’s optimal abilities, it is critical that great detail
is paid to its process of induction. As such, the heterodimerization of CB1 and
5-HT2A receptors must be assessed to understand the mode of regulation
that underlies the analgesic and amnesiac consequences of THC induced
activation. The proposed study aims to determine the time point of
heterodimerization and will subsequently allow for determination of its
location within the cells. As a result, future steps will encompass further
assessment of the proteins present within the location of dimerization to
determine the regulatory mechanism. Determination of the regulatory
mechanism will provide insight to targets and modes of inhibition for
dimerization disruption to isolate the analgesic properties of the THC
d) The same procedure will be conducted on on three negative control activated CB1 receptor.
studies where Genome editing to modify cells will be accomplished by
using ThermoFisher Scientific CRISPR-Cas9 Kit.
• Control 1: CB1 receptor knocked out Acknowledgements
• Control 2: 5-HT2A receptor knocked out
• Control 3: Both receptors knocked out Bai, M. (2004). Dimerization of G-protein-coupled receptors: roles in signal transduction. Cellular signalling, 16(2), 175-186.
e) Quantification of histidine-tagged ERK in test and controls. Bhattacharyya, S., Egerton, A., Kim, E., Rosso, L., Barros, D. R., Hammers, A., ... & McGuire, P. (2017). Acute induction of anxiety in humans
• Histidine-tag will be added using ThermoFisher Scientific CRISPR-
by delta-9-tetrahydrocannabinol related to amygdalar cannabinoid-1 (CB1) receptors. Scientific Reports, 7(1), 15025.
Cas9 Kit.
Bombardi, C., & Di Giovanni, G. (2013). Functional anatomy of 5-HT 2A receptors in the amygdala and hippocampal complex: relevance to
memory functions. Experimental brain research, 230(4), 427-439.
• Isolation of His-tagged ERK using Nickel column chromatography. Busquets-Garcia, A., Gomis-González, M., Salgado-Mendialdúa, V., Galera-López, L., Puighermanal, E., Martín-García, E., ... & Ozaita, A.
(2018). Hippocampal Protein Kinase C Signaling Mediates the Short-Term Memory Impairment Induced by Delta9-
Tetrahydrocannabinol. Neuropsychopharmacology, 43(5), 1021.
Elikottil, J., Gupta, P., & Gupta, K. (2009). The analgesic potential of cannabinoids. Journal of opioid management, 5(6), 341.
Howlett, A. C., Blume, L. C., & Dalton, G. D. (2010). CB1 cannabinoid receptors and their associated proteins. Current medicinal chemistry,
CB1 - YFP
17(14), 1382-1393.
Kaminski, C. F., Rees, E. J., & Schierle, G. S. K. (2014). A quantitative protocol for intensity-based live cell FRET imaging. In Fluorescence
Spectroscopy and Microscopy (pp. 445-454). Humana Press, Totowa, NJ.
Laprairie, R. B., Bagher, A. M., & Denovan-Wright, E. M. (2017). Cannabinoid receptor ligand bias: implications in the central nervous system.
Current opinion in pharmacology, 32, 32-43.
Milligan, G. (2007). G protein-coupled receptor dimerisation: molecular basis and relevance to function. Biochimica et Biophysica Acta (BBA)-
Genetic Code Expansion Biomembranes, 1768(4), 825-835.
5-HT2A Seibenhener, M. L., & Wooten, M. W. (2012). Isolation and culture of hippocampal neurons from prenatal mice. Journal of visualized
experiments: JoVE, (65).
Viñals, X., Moreno, E., Lanfumey, L., Cordomí, A., Pastor, A., de La Torre, R., ... & Lluís, C. (2015). Cognitive impairment induced by delta9-
tetrahydrocannabinol occurs through heteromers between cannabinoid CB1 and serotonin 5-HT2A receptors. PLoS biology, 13(7),
e1002194.
Walker, J. M., & Hohmann, A. G. (2005). Cannabinoid mechanisms of pain suppression. In Cannabinoids (pp. 509-554). Springer, Berlin,
Heidelberg.