G Protein Coupled Receptors

Let’s start with the basics. The cell membrane is composed of phospholipids. Each phospholipid has a water loving head group and two acyl chains that are hydrophobic… they hate water. The inside of the cell is separated from the outside world by two layers of these phospholipids. These hydrophobic acyl chains keep the outside water world from mixing with the inside water world. Oil and water do not mix. Transmembrane proteins are there to inform the inside of the cell what is going on in the outside world without actually having to get mixed up with it.

Figure 1 G-protein coupled receptors communicate the ongoing of the environment outside the cell with the enzymes inside the cell. Part 1 is release of the α and βγ subunits from their inactive receptor bound complexes.

G-protein coupled receptors are named after the hetero-trimeric G proteins that bind to the last intracelular loop. Hetero means different and tri means three. They are given Greek names alpha, beta, gamma. The α subunit binds to the receptor, but only if it binding the small molecule GDP. The βγ subunits travel as a pair. The βγ binds to α -GDP. When an agonist binds to the outside of the GPCR, the GDP leaves the α sub unit only to be replaced by GTP. Gα-GTP dissociates from the receptor to perform receptor directed functions. βγ also binds other proteins inside the cell signalling them to perform their functions.

Figure 2. GTP and GDP explained. The orange P surrounded by four red Os is a phosphate.

The βγ subunit

Figure 1 depicts the βγ sub unit hanging out at the cell membrane in the active form. The βγ sub unit modulates other cellular proteins [1]

  • The inwardly rectifying potassium (GIRK) channel is activated by the βγ sub unit. This channel is also modulated by phosphatidyl inositol 4,5-bisphosphate PIP2 {1]
  • The dopamine reuptake transporter is under GPCR control via the βγ subunit.[1] Dopamine receptors are GPCR.
  • The transient potential M3 (TRPM3) ion channels are inhibited by the βγ subunit. [1]
  • Phosphoinositide 3 kinase β (PI3Kβ) mediates modulates the phosphorylation of inositol head groups of membrane phospho lipids important in cell signalling. This enzyme is modulated by the βγ subunit. [1] PI3Kβ plays a role in producing PIP2 by phosphorylating the inositol head group.

The review also discusses how the βγ sub unit participates in the recycling of heterotrimeric G protein / receptor complexes. In depth structural analyses are given for the interested reader. [1]

α Subunit Flavors

There are at least four types of alpha subunit. We will only look in depth on the inhibitory and stimulatory flavors as these are the ones that interact with cannabinoid receptors.

GsαGiα
serotonin receptors 5-HT4 &5-HT7    Serotonin receptors 5-HT1 & 5-HT5
    Adenosine receptor A2a and A2b    Adenosine A1 & A3 receptors
    β-adrenergic receptors types β1-3,     Adrenergic receptors α2A-C
    Calcitonin receptor    Calcium-sensing receptor
    Cannabinoid receptor CB2    Cannabinoid receptors (CB1 and CB2)
    Dopamine receptors D1 and D5    Dopamine receptors D2, D3, D4
    Glutamate  mGlu3-8 
    Histamine H2 receptor    Histamine H3 & H4 receptors
    Melatonin MT1-3 receptors
    Opioid δ, κ, μ, & nociceptin receptors
    Olfactory receptors
    Prostaglandin receptor D2 and I2    Prostaglandin receptors EP1, EP3, FP, & TP 
   Trace amine-associated receptor 1    Trace amine-associated receptor 8
Table 1 Receptors that interact with alphas and alphai subunits. These lists ar edited from those appearing on Wikipedia. Examples of those sharing a common ligand are shown. Othres that might have an entourage effect such as the olfactory receptors and melatonin receptors are shown.

Missing from this list is G α q subunit. Golf-α is expressed in the olfactory bulb as well the striatum. It functions as a Gs-α subunit.

The adenosine A2A receptor, a target of limonene

The adenosine A2A receptor is also a G-protein coupled receptor (GPCR). In a cell culture system limonene binds to the A2A receptor, increases cAMP production, activates protein kinase A which phosphorylates down stream targets. [2]. In 2021 a Korean group published a mouse study in which mice were injected with limonene (10 mg/kg) or a saline control. [3] Behavior studies showed less anxiety in the treatment group. Less anxiety was defined as increased loco-motor activity and open arm preference in an elevated maze test. [3] Methamphetamine (1mg/kg) was used for comparison. Treatments were given 30 minutes before tests. [3]

  • Meth mice covered about 500 m in 5 min,
  • control mice less than 200m, and
  • limonene mice maybe a little over 200 m. [3]

The GABAA chloride channel agonist Diazepam made exploration of an open arm in a maze more attractive in terms of time spent, time to entry, and so on. Limonene had less of an effect in these regards. [3] These authors looked at protein levels of an enzyme involved in dopamine synthesis tyrosine hydroxylase. Levels of dopamine may be increased by preventing its release by glutamate. Levels of glutamate decarboxylase also increased. {3] Gene transcription is controlled by the cAMP element response binding protein (CREB) phosphorylation by PKC. This too was increased by limonene. [3] Finally, dopamine levels themselves were increased. [3] Wikipedia authors broach the surface of how really complexity of connections in the striatum. In looking at CB1 adenosine A2A receptor interactions, we need to remember that dopamine receptor and A2A interactions also modulate connections in our brains. Take note that it is the inter neurons that express tyrosine hydrolase.

Note: These contradictions are very real and confusing: The runners’ high annandamide binds to a Giα coupled receptor CB1 and reduces anxiety. Limonene binds to a Gsα coupled receptor adenosine A2A and reduces anxiety. In just a single cell the contradictory roles of these receptors is like the fine control one gets from driving with one foot on the brake and the other on the accelerator.

Figure 3. G-protein coupled receptors operate with the fine control one gets from driving with one foot on the brake and another foot on the accelerator. The CB1 receptor couples with G α i while the andenosine A2A receptor couples with G α s. One inhibits adenylyl cylase, the other stimulates anylyl cyclase. This enzyme converts ATP to cyclic AMP (cADP). cAMP activates protein kinase A. Protein kinase A attaches a phosphate group to a serines, threonines, or tyrosines of a myriad of proteins that have diverse functions in the cell.

The heart and sole of G-protein coupled protein signalling is cAMP activated protein kinase A (PKA). This link gives an extensive overview of pathways and disease states impacted by PKA. PKA signalling impacts the following pathways: gene expression and nuclear dynamics, cardiovascular function, metabolism, neuronal receptors and ion channels, and cytoskeletal dynamics.

The adenosine & anandamide entourage

The adenosine A2A and CB1 receptor interactions have been well studied in the medium-sized spiny neurons of the striatum. [4] These neurons integrate signals from GABA and glutamate releasing afferent. MSN make up 95% of the population of the stiatrum. [4] There re two main classes of MSN:

Endogenous opioid enkephalin-ergic MSNs

connect the striatum with the globus pallidus, which regulates voluntary movements.

  1. dopamine D2
  2. adenosine A2A
  3. Adenosine A1 receptors may also be found.
Analgesic endogenous opioid dynorphinergic MSNs

connect the striatum with the rewards and movement center the substantia nigra.

  1. Dopamine D1
  2. adenosine A1 but NOT A2A receptors ,
  3. glutamate receptors as well, but these are glutamate gated Ca2+ channels rather than GPCR.
Figure 4 The interplay between CB1 and Adenosine A2A receptors in medium-sized spiny neurons in the striatum. Parts of this image are adapted from reference [4].
  1. When the endocannabinoid tone is high and the adenosine tone is low, the CB1 A2AR dimer acts as a brake because no adenosine is binding to A2AR. Adenosine is binding to A1R, another Gi coupler but not to the lower affinity A2AR. Two sets of brakes are being applied to adenylyl cyclase. [4]
  2. When both the endocannabinoid and adenosine tones are low, only the A1R has ligand bound and the brake is on andenylyl cyclase. [4]
  3. When both endocannabinoid and adenosine tones are high, CB1/A2AR acts as a brake, A2AR/CB1/A2AR acts as an accelerator, and A1R/A2AR doesn’t to anything. [4]
  4. When the adenosine tone is high and endocannabinoid tone is low, noting happens with the CB1/A2AR dimer. The A2AR/A2AR is accelerator is on, and the A2AR/A1R is doing nothing. [4]

For the interested reader reference [5] describes some of the actual experiments that allowed the authors [4] to come to their conclusions.

So far, still a gross simplification of the striatum…

In 2004 a group from Rome, Italy used immunohistochemistry and confocal microscopy to co-localize CB1 with various subsets of interneurons of the rat striatum. [6]

  • Calbinding-28 striatum matrix projections
  • Choline acetyl transfersase ChAT striatum cholinergic interneurons
  • parvalbumin GABAergic/parvalbuminergic striatum interneurons
  • calrecticulin, neuronal nitric oxide synthase, neuropeptide Y-somatostatin… interneurons
Figure 5 Unterneurons add an additional level of fine tuning A. A interneuron between glutamergic cortical neurons projecting onto the striatum. B. Exerts from Figure 4 D-F from ref {6] C. Table 4 from ref [6]

CB1 receptors on striatum inter neuron brakes starts to address the paradox of why limonene and anandamide can bind to opposing receptors in an individual neuron yet both decrease anxiety in rat models.

The THC/limonene or caffeine anandamide entourage…

Some cannabis users might like strains that contain limonene because it puts the brakes on the high making it a bit more subdued. Caffeine is a natural, plant based medicine, antagonist of the adenosine A2A receptor. Antagonist means that it antagonizes the action of adenosine and takes things into the direction of CB1 activation. Wikipedia authors do a good job of describing the action of caffeine in the striatum. Caffeine action on ryanodine receptors controlling calcium stores in muscle is given a good mention.

Figure 56 A collage of images promoting coffee and running/cycling.

Take a look at coffee shops where runners and bicyclists hang out after a good workout. Don’t they seem kind of happy and high even though they are probably worn out? Could it be that caffeine enhances the endocannabinoid high?

How do these weekend athletes monitor their blending of natural CB1 agonists and a plant based adenosine A2A antagonist? By what feels good? How would a physician advise a patient in use of a strain of cannabis with THC and limonene as a dominant terpene?

References

  1. Smrcka, A. V., & Fisher, I. (2019). G-protein βγ subunits as multi-functional scaffolds and transducers in G-protein-coupled receptor signaling. Cellular and molecular life sciences : CMLS, 76(22), 4447–4459. PMC free article
  2. Park HM, Lee JH, Yaoyao J, Jun HJ, Lee SJ. Limonene, a natural cyclic terpene, is an agonistic ligand for adenosine A(2A) receptors. Biochem Biophys Res Commun. 2011 Jan 7;404(1):345-8.
  3. Song Y, Seo S, Lamichhane S, Seo J, Hong JT, Cha HJ, Yun J. 92021)Limonene has anti-anxiety activity via adenosine A2A receptor-mediated regulation of dopaminergic and GABAergic neuronal function in the striatum. Phytomedicine. 2021 Mar;83:153474. PMC free article
  4. Ferré, S., Lluís, C., Justinova, Z., Quiroz, C., Orru, M., Navarro, G., Canela, E. I., Franco, R., & Goldberg, S. R. (2010). Adenosine-cannabinoid receptor interactions. Implications for striatal function. British journal of pharmacology, 160(3), 443–453. PMC free article
  5. Ferreira, S. G., Gonçalves, F. Q., Marques, J. M., Tomé, Â. R., Rodrigues, R. J., Nunes-Correia, I., Ledent, C., Harkany, T., Venance, L., Cunha, R. A., & Köfalvi, A. (2015). Presynaptic adenosine A2A receptors dampen cannabinoid CB1 receptor-mediated inhibition of corticostriatal glutamatergic transmission. British journal of pharmacology, 172(4), 1074–1086. PMC free article
  6. Fusco FR, Martorana A, Giampà C, De March Z, Farini D, D’Angelo V, Sancesario G, Bernardi G. (2004) Immunolocalization of CB1 receptor in rat striatal neurons: a confocal microscopy study.
  7. Synapse. 2004 Sep 1;53(3):159-67.

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