Cannalingo, Part I: A Guided Tour Through the Influence of Protein Structure and Function on Cannabinoid Signaling

Have you ever wondered why some people are more sensitive to the effects of cannabinoids than others? Factors such as dosing, metabolic rates, and weight influence these differences. But there is a more complex bustling of tiny, molecular processes that specifically dictate these differences in predictable ways. To gain an understanding of the busy, massive, yet focused interactions that dictate this process, one must wade into the sea of organic molecules that constantly feel and touch one another to distribute, accept, and receive messages to influence cellular communication within the body. I hope to begin this journey with you, and together we can gain an appreciation for which proteins decide our response to cannabinoids, and investigate how variants in our population may influence reactions to the cannabis experience. We begin with an in-depth exploration of the structure, function, and variation of cannabinoid receptor 1 (CB1R).

Why Should You Explore This Molecule?

The influence of major molecular products of cannabinoid producing plants mediate their biological effects through two G-protein coupled receptors of the cannabinoid receptor family (CB1R and CB2R). CB1R has garnered the most molecular, medicinal, and research attention. This receptor is the gatekeeper for many of the effects felt from cannabis consumption and will serve as our entry point into this molecular journey.

How Will This Molecule Be Explored?

A close look will be taken at the structure of CB1R and its interactions with cannabinoids on the exterior of the cell. Then, a ride through the induced cellular signaling will be taken to consider how the cell changes its behavior to elicit the cannabis experience. During this journey, we will meet some of the other molecules that play a role in that experience, to be studied further in the series. Surprisingly, no variants have been characterized for CB1R that alter the structure of the receptor. There have been seven “silent” mutations discovered in humans. These variants cause no change in the building blocks that assemble the receptor. However, there have been duplications of the gene that encode the receptor identified in a few individuals. Therefore, we must spend some time to unravel the mysteries associated with having more copies of the gene.

What Will You Do with This Information?

Perhaps you are working in a drug discovery laboratory and this will help you infer decisions on your project. Maybe you are interested in learning about the molecular messages, and how cannabinoids communicate. Or maybe you are simply curious. Several gene sequencing products now exist for genealogy and medicinal purposes. Here is where consumers can get real use out of such services. How powerful could it be to know whether your patients or customers will respond to a specific cannabinoid and at which doses? Grasping the content of this series will open the door to discovering the secret messages of the cellular signaling, or “cannalingo,” happening within our bodies.

It has been more than 5000 years since the first evidence of cannabinoid use was documented in China for the treatment of pain and cramps (1). Since that time, cannabinoids have been used to combat a myriad of conditions including blocking pain reception, inflammation, reducing the severity of epileptic fits, prevention of nausea and vomiting, as well as recreational use. Much is left to be understood about our individual responses to cannabinoid use and exposure. Summarizing what is currently understood of the structure and expression density of CB1R will contribute to the appreciation of the differences in reactions individuals have when exposed to cannabinoids. To properly explore the attributes of CB1R expression and function in cannabinoid signaling, a brief introduction to the central dogma of molecular biology is in store.

All but our mature red blood and cornified cells (such as hair and skin) carry a copy of our genome in the nucleus of our cells. Our genomes are comprised of approximately 30,000 unique genes. Many of these genes provide instructions for making the estimated 80,000 to 400,000 different proteins, and these molecules perform much of the work that keeps us alive and well. The interaction of these proteins provides us with our phenotype; our individual characteristics including appearance, behaviors, and cellular functions. While humans may theoretically express up 400,000 different proteins, protein expression is based on cell type. There are more than 250 different types of cells that assemble to comprise and support our bodies. This creates a lot of variability in protein expression between cells. Not all cells express the same proteins, which is what differentiates one cell type from the other. Our genomes are comprised of more than 3,000,000,000 (yes, that’s 3 billion, with a b) pairs of nucleotides, the building blocks of our deoxyribonucleic acid (DNA). These nucleotides (adenine, guanine, cytosine, and thymine) are organized into 23 pairs of chromosomes. We inherit each set of chromosomes 1 to 23 from our biological parents.

When cells receive a queue from themselves or the environment they reside, a series of messages are sent within the cell, many of which are sent to the nucleus, which harbors our DNA. Messages received by the nucleus instruct changes in the expression of genes required to change the behavior of the cell. For example, these messages can stimulate mental and physical relaxation, induce hunger, or trigger sleepiness. To enact these behavioral changes, many macromolecules (nucleic acids, proteins, carbohydrates, and lipids) will cooperate to relax sections of our genomes, enabling molecular machinery to gain access to the genetic sequence of the DNA, and producing copies of small sections of the genome, which correspond to protein-encoding regions. This leads to the transcription of a temporary, unstable copy of the gene in the form of messenger ribonucleic acid (mRNA). After some processing for maturation, this mRNA leaves the nucleus and works with other molecular machinery in the cytoplasm of the cell to coordinate translating the mRNA into the sequence of amino acids, which are the building blocks that comprise the CB1R protein. Think of this as translating a sentence from one language to another using a different alphabet. This protein will often undergo post-translational modifications that will enable the protein to perform its intended function. This induced expression and subsequent interaction with cannabinoids will initiate the behavioral change intended by the messages received by the cell.

While the order of genes on each of our chromosomes is similar within our population, the sequence of the nucleotides that comprise each chromosome varies by ~0.1% between individuals. This is the source of variation that makes each of us unique. These variations result because of mutations, revisions, to the sequence of nucleotides within a given gene. A great metaphor for this can be seen in the early episodes of the most recent Marvel Cinema Universe (MCU) escape, Loki. The main character of the series, Loki, uses magic to escape from captivity by traveling through time. He is then considered a “Variant,” and is charged with the crime of wreaking havoc across the Sacred Timeline. Many variants may exist for a given gene. This causes the variation we see among humans. Some of these variants produce new traits. Others will result in unintended consequences for the functional component of that gene (for example, the resulting protein), while others may cause no change in protein function at all. The sequence of amino acids dictates the structure the protein will fold into. It’s the structure of the protein that dictates its function. Changes to this sequence can result in functional changes.

Once assembled, proteins can be shuttled throughout the cell using roadways and transport molecules comprised of other interacting macromolecules. Some proteins will remain within the cell (intracellular), either in the aqueous medium of the cytoplasm or organelles within the cell. They become embedded within a membrane of an organelle or plasma membrane or are secreted from the cell to invoke changes in the extracellular matrix. CB1R is expressed in membrane structures (Figure 1).

The endocannabinoid system is a series of receptors, enzymes, and endocannabinoids that cooperate to induce responses to cannabinoid exposure. We will explore variations of several key players involved in the endocannabinoid system throughout this series. Cannabinoid receptor 1 (CB1R) is one of two cannabinoid receptors that contribute to the endocannabinoid system in humans. CNR1, the gene that encodes for CB1R, is located on chromosome 6 (Figure 2). It is comprised of 24,486 nucleotides organized into 8 exons and 6 introns. During the process of transcription, the introns are spliced, or cut, out of the mRNA sequence and the exons are joined. The exons can be spliced together to form one of two different isoforms (versions) of mRNA that will provide the instructions for assembling the specific sequence of amino acids that comprise either version of the protein. The most common form, CB1R, is comprised of 472 amino acids, while a truncated version, CB1Rb, is 33 amino acids shorter at 439 amino acids. 

A member of the G protein coupled receptor family, CB1R associates with G protein α subunits including Gi α-1/GNAI3 and Go α-GNAO1 (2). The function of CB1R is wildly cell dependent and varies between cell types. While significant research remains to be completed to gain a greater appreciation for the vast functions of CB1R, efforts have been made to begin to piece together the beginnings of a story. CB1R has been demonstrated to participate in executive, emotional, memory, and reward processing signals upon stimulation by cannabinoids (3). CB1R is prominently expressed in the cells of the central nervous system, and to a lesser extent in fat cells, placental, adrenal gland, and other cell types (Figure 3). 

The receptor is embedded in the plasma membrane that surrounds the cell, separating the cell from its environment and other cells, as well as endosome and lysosome membranes and the outer mitochondrial membrane. CB1Rs expressed on the cell membrane are suspected to play unique roles depending upon which other receptors on the cell it engages with upon stimulation. Unlike traditional G coupled protein receptors, CB1R inhibits the formation of cyclic adenosine monophosphate (cAMP), a molecule significant to amplifying cellular signals involved in metabolic pathways that contribute to increasing access to cellular fuel reserves and ATP production, among other things. Rather CB1R signaling contributes to a reduction of intracellular calcium levels and increased MAPK signaling. Depending upon other contributing proteins in the specific cell type, MAPK activity can result in cell survival or death. Conversely, CB1R expressed in lysosome membranes initiates an increase of cellular calcium levels and permeability. Mitochondrial CB1R signaling results in decreased mitochondrial metabolism (respiration), and may or may not serve a protective role for the powerhouse of our cells.

Aside from serving a significant role in cannabinoid signaling, CB1R has been investigated as a potential therapeutic target for neuropsychological disorders and neurodegenerative diseases. The studies summarized above lend evidence to further research this possible benefit. CB1R has been demonstrated to modulate neurotransmission and mediate neuroprotection against excitotoxity, an overstimulation of neurons which often results in neuronal death. In fact, CB1R has even been suggested to be upregulated in neurodegenerative disease models, which is believed to play a role in compensating for aberrant neuronal signaling characteristic of such conditions. Alzheimer’s disease models have demonstrated a possible CB1R activation preventative strategy in combating the toxicity induced by amyloid-β accumulation, whereas a reduction of CB1R expression has been suggested to occur in progressive neurodegenerative diseases such as Huntington’s disease (summarized in review by Zou and colleauges [4]). 

The CB1R structure is comprised of seven alpha helices assembled to produce a transmembrane portion that spans the membrane, an extracellular domain that interacts with delta-9-tetrahydrocannabinol, endocannabinoids 2-arachidonoylglycerol (AG-2), and anadamide (AEA), and a highly disordered intracellular domain that interacts with cytoplasmic components of signaling (Figure 4). Upon stimulation by cannabinoids, CB1R will initiate a cascade of reactions in the cell (or organelle) that lead to alterations of mood and cognition.

Expression of full length CB1R dominates in the brain and skeletal muscle, with increased expression density in presynaptic terminals of neurons (5,6), whereas the truncated version is abundant in liver and pancreas (7). While amino acids 2–23 are required for transporting the protein to the mitochondria, amino acids 22–54 are missing from CB1Rb. The functional differences between the two isoforms has yet to be fully elucidated. Fortunately, these residues are not thought to bind to cannabinoids, so the truncation is not suspected to significantly disrupt binding and subsequent cell signaling effects. Rather, the hydrophobic pocket created at the apex of interacting alpha helices is maintained. Aside from these two isoforms, seven variants have been characterized in the literature (2) (high-quality and freely accessible set of protein sequences annotated with functional information). In this article, we describe significant updates that we have made over the last two years to the resource. The number of sequences in UniProtKB has risen to approximately 190 million, despite continued work to reduce sequence redundancy at the proteome level. We have adopted new methods of assessing proteome completeness and quality. We continue to extract detailed annotations from the literature to add to reviewed entries and supplement these in unreviewed entries with annotations provided by automated systems such as the newly implemented Association-Rule-Based Annotator (ARBA). These variants are not as chaotic as our earlier described Loki variant. All seven variants are silent mutations that do not result in changes to the amino acid sequence for the protein. While the DNA sequence may differ among these variants, the transcribed amino acid sequence and resulting protein structure are unchanged. Thus, CB1R is a highly conserved protein in our population and across species.

What’s interesting is rather than demonstrating variants of protein structure and function among the population, entire gene duplication events have been characterized. Several duplications of sections of chromosome 6 have been described in the literature (8). Sometimes this duplication of genetic material includes one to three extra copies of the section that encodes for CB1R. These duplications may cause increased expression of the receptor. An increased expression of the receptor may lead to an amplified response to cannabinoid signaling. Studies to investigate this have yet to be completed, and further research is required to test this hypothesis among individuals with chromosome 6 duplications. The implications of expression and response to cannabinoid exposure would be an interesting correlation to assess among the population.

Many clues regarding the role CB1R plays in our responses to cannabinoid exposure have been revealed. However, this is only the beginning of fully elucidating the molecular and, subsequently, behavioral consequences of cannabinoid use. CB1R forms complexes with several other receptors embedded within membranes. These unique pairings result in more complex and novel signaling mechanisms. Further research is needed to decode these pairings, and better understand the role CB1R plays in cannabinoid signaling in specific cells. A consideration of expression density by cell type, correlated with CNR1 gene duplication should follow suit. In addition, the diverse expression of CB1R within the cell provides rationale for investigating mechanisms that regulate cannabinoid permeability through the plasma membrane to confer intracellular interactions. The versatility of the signaling pathways induced by CB1R stimulation and its widespread expression throughout the body serves as justification for supporting further evaluation of this protein as a target for various diseases, conditions, and simply better understanding consumer needs. Further consideration of the natural variants among other proteins significant to the endocannabinoid system will improve our grasp of the role cannabinoids have in their potential pharmacological diversity and expanded recreational use. To paraphrase Loki’s handler Mobius, studying these variants among the endocannabinoid system are the key to understanding the molecular consequences cannabinoid use.

References

1. R. Mechoulam, Cannabinoids as Therapeutic Agents. 1st ed. (Chapman and Hall/CRC, Boca Raton, Florida, 1986) pp 1-20.
2. The UniProt Consortium. UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Research. 49(D1), D480-D489 (2021).
3. J. Wu, Cannabis, Acta Pharmacol Sin. 40(3), 297-299 (2019).
4. S. Zou and U. Kumar, Int. J. Mol. Sci. 19(3) 833 https://doi.org/10.3390/ijms19030833 (2018).
5. I. Katona, B. Sperlágh, and A. Sík, et al. J. Neurosci. 19(11), 4544–4558 (1999).
6. K. Tsou, S. Brown, M.C. Sañudo-Peña, K. Mackie, and J.M. Walker, Neuroscience 83(2), 393–411 (1998).
7. I. González-Mariscal, S.M. Krzysik-Walker, and M.E. Doyle, et al. Sci Rep. 6, 33302 (2016).
8. M.J. Landrum, J.M. Lee, and M. Benson, et al. Nucleic Acids Res. 46(D1), D1062-D1067 (2018).
9. Gene [Internet]. Gene ID 1268, CNR1 cannabinoid receptor 1 [Homo sapiens (human)]. Published online June 6, 2021. Accessed June 17, 2021. https://www.ncbi.nlm.nih.gov/gene/1268.
10. Z. Shao, J. Yin, and K. Chapman, et al. Nature 540(7634), 602-606 (2016).

About the Author

AUDREY SHOR is an Associate Professor of Biology at Saint Leo University in Saint Leo, Florida and a cofounder and Chief Science Officer at Decarb Factor in Denver, Colorado.

Direct correspondence to:
audrey@decarbfactor.com

How to Cite this Article

A. Shor, Cannabis Science and Technology 4(7), 44-54 (2021).