*Evolutionary Origins of Endocannabinoid System



Elphick & Egertová 2005

Handb Exp Pharmacol. 2005;(168):283-97.


The phylogenetic distribution and evolutionary origins of endocannabinoid signalling.

Elphick MR, Egertová M.

School of Biological Sciences, Queen Mary, University of London, London E1 4NS, UK. M.R.Elphick@quml.ac.uk

The endocannabinoid signalling system in mammals comprises several molecular components,

….including cannabinoid receptors (e.g. CB1, CB2),

….putative endogenous ligands for these receptors [e.g. anandamide, 2-arachidonoylglycerol (2-AG)]

….and enzymes involved in the biosynthesis and inactivation of anandamide (e.g. NAPE-PLD, FAAH) and 2-AG (e.g. DAG lipase, MGL).

In this review we examine the occurrence of these molecules in non-mammalian organisms (in particular, animals and plants) by surveying published data and by basic local alignment search tool (BLAST) analysis of the GenBank database and of genomic sequence data from several vertebrate and invertebrate species.

We conclude that the ability of cells to synthesise molecules that are categorized as “endocannabinoids” in mammals

is an evolutionarily ancient phenomenon

that may date back to the unicellular common ancestor

of animals and plants.

……….However, exploitation of these molecules for intercellular signalling may have occurred independently in different lineages during the evolution of the eukaryotes.

The CB1- and CB2-type receptors that mediate effects of endocannabinoids in mammals occur throughout the vertebrates,

………and an orthologue of vertebrate cannabinoid receptors was recently identified in the deuterostomian invertebrate Ciona intestinalis (CiCBR).

However, orthologues of the vertebrate cannabinoid receptors are not found in protostomian invertebrates (e.g. Drosophila, Caenorhabditis elegans). Therefore, it is likely that a CB1/CB2-type cannabinoid receptor originated in a deuterostomian invertebrate. This phylogenetic information provides a basis for exploitation of selected non-mammalian organisms as model systems for research on endocannabinoid signalling.

See also:

Philos Trans R Soc Lond B Biol Sci. 2001 Mar 29;356(1407):381-408.


The neurobiology and evolution of cannabinoid signalling.

Elphick MR, Egertová M.

School of Biological Sciences, Queen Mary, University of London, London E1 4NS, UK. m.r.elphick@@qmw.ac.uk


McPartland, Norris & Kilpatrick 2007b

Gene. 2007 Aug 1;397(1-2):126-35. Epub 2007 Apr 25.


Coevolution between cannabinoid receptors and endocannabinoid ligands.

McPartland JM, Norris RW, Kilpatrick CW.

GW Pharmaceuticals, Middlebury, VT 05753, USA. mcpruitt@verizon.net

Genes for receptors and ligands must coevolve to maintain coordinated gene expression and binding affinities. Researchers have debated whether anandamide or 2-arachidonyl glycerol (2-AG) is a more “intrinsic” ligand of cannabinoid receptors.

We addressed this debate with a coevolutionary analysis, by examining genes for CB1, CB2, and ten genes that encode ligand metabolic enzymes: abhydrolase domain containing 4 protein, cyclooxygenase 2, diacylglycerol lipase paralogs (DAGLalpha, DAGLbeta), fatty acid amide hydrolase paralogs (FAAH1, FAAH2), monoglyceride lipase, N-acylethanolamine acid amidase, NAPE-selective phospholipase D, and protein tyrosine phosphatase non-receptor type 22.

Gene trees (cladograms) of CB1, CB2, and ligand enzymes were obtained by searching for orthologs (tBLASTn) in the genomes of nine phylogenetically diverse species, aligning ortholog sequences with ClustalX, and applying Bayesian analysis (MrBayes). Mirrored cladograms provided evidence of coevolution (i.e., parallel cladogenesis). ….

we propose that cannabinoid receptors

initially coevolved with a fatty acid ester ligand (akin to 2-AG)

in ancestral metazoans,

and affinity for fatty acid ethanolamide ligands (e.g., AEA)

evolved thereafter.


Wildman et al 2003

Proc Natl Acad Sci U S A. 2003 Jun 10;100(12):7181-8. Epub 2003 May 23.


Implications of natural selection in shaping 99.4% nonsynonymous DNA identity between humans and chimpanzees: enlarging genus Homo.

Wildman DE, Uddin M, Liu G, Grossman LI, Goodman M.

Center for Molecular Medicine and Genetics and Department of Anatomy and Cell Biology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201, USA.

What do functionally important DNA sites, those scrutinized and shaped by natural selection, tell us about the place of humans in evolution? Here we compare approximately 90 kb of coding DNA nucleotide sequence from 97 human genes to their sequenced chimpanzee counterparts and to available sequenced gorilla, orangutan, and Old World monkey counterparts, and, on a more limited basis, to mouse.

The nonsynonymous changes (functionally important), like synonymous changes (functionally much less important), show chimpanzees and humans to be most closely related, sharing 99.4% identity at nonsynonymous sites and 98.4% at synonymous sites.

On a time scale,

the coding DNA divergencies separate the human-chimpanzee clade

from the gorilla clade at between 6 and 7 million years ago

and place the most recent common ancestor of humans and chimpanzees

at between 5 and 6 million years ago.

The evolutionary rate of coding DNA in the catarrhine clade (Old World monkey and ape, including human) is much slower than in the lineage to mouse. Among the genes examined, 30 show evidence of positive selection during descent of catarrhines. Nonsynonymous substitutions by themselves, in this subset of positively selected genes, group

humans and chimpanzees closest to each other

and have chimpanzees diverge about as much from the common human-chimpanzee ancestor as humans do.

This functional DNA evidence supports two previously offered taxonomic proposals:

family Hominidae should include all extant apes; and

genus Homo should include three extant species and two subgenera,

Homo (Homo) sapiens (humankind),

Homo (Pan) troglodytes (common chimpanzee), and

Homo (Pan) paniscus (bonobo chimpanzee).

See also:

Proc Natl Acad Sci U S A. 2008 Mar 4;105(9):3215-20. Epub 2008 Feb 27.


Distinct genomic signatures of adaptation in pre- and postnatal environments during human evolution.

Uddin M, Goodman M, Erez O, Romero R, Liu G, Islam M, Opazo JC, Sherwood CC, Grossman LI, Wildman DE.

Center for Molecular Medicine and Genetics and Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, MI 48201, USA.


Elphick, Satou & Satoh 2003

Abstract – UK

The G-protein coupled cannabinoid receptors CB(1) and CB(2) are activated by Delta(9)-tetrahydrocannabinol, the psychoactive ingredient of cannabis, and mediate physiological effects of endogenous cannabinoids (‘endocannabinoids’).

CB(1) genes have been identified in mammals, birds, amphibians and fish, whilst CB(2) genes have been identified in mammals and in the puffer fish Fugu rubripes. Therefore, both CB(1) and CB(2) receptors probably occur throughout the vertebrates.

However, cannabinoid receptor genes have yet to be identified in any invertebrate species and the evolutionary origin of cannabinoid receptors is unknown. Here we report the identification of CiCBR, a G-protein coupled receptor in a deuterostomian invertebrate – the urochordate Ciona intestinalis – that is orthologous to vertebrate cannabinoid receptors. The CiCBR cDNA encodes a protein with a predicted length (423 amino-acids) that is the intermediate of human CB(1) (472 amino-acids) and human CB(2) (360-amino-acid) receptors.

Interestingly, the protein-coding region of the CiCBR gene is interrupted by seven introns, unlike in vertebrate cannabinoid receptor genes where the protein-coding region is typically intronless. Phylogenetic analysis revealed that CiCBR forms a clade with vertebrate cannabinoid receptors

………. but is positioned outside the CB(1) and CB(2) clades of a phylogenetic tree,

………indicating that the common ancestor of CiCBR and vertebrate cannabinoid receptors predates a gene (genome) duplication event that gave rise to CB(1)- and CB(2)-type receptors in vertebrates.

Importantly, the discovery of CiCBR and the absence of orthologues of CiCBR in protostomian invertebrates such as Drosophila melanogaster and Caenorhabditis elegans indicate that

the ancestor of vertebrate CB(1) and CB(2) cannabinoid receptors originated in a deuterostomian invertebrate.


Matais et al 2002

Eur J Biochem. 2002 Aug;269(15):3771-8.


Presence and regulation of the endocannabinoid system in human dendritic cells.

Matias I, Pochard P, Orlando P, Salzet M, Pestel J, Di Marzo V.

Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Comprensorio Olivetti, Pozzuoli (Napoli), Italy.

Cannabinoid receptors and their endogenous ligands, the endocannabinoids,

……..have been detected in several blood immune cells, including monocytes/macrophages, basophils and lymphocytes.

However, their presence in dendritic cells, which play a key role in the initiation and development of the immune response, has never been investigated. Here we have analyzed human dendritic cells for the presence of the endocannabinoids, anandamide and 2-arachidonoylglycerol (2-AG), the cannabinoid CB1 and CB2 receptors, and one of the enzymes mostly responsible for endocannabinoid hydrolysis, the fatty acid amide hydrolase (FAAH).

By using a very sensitive liquid chromatography-atmospheric pressure chemical ionization-mass spectrometric (LC-APCI-MS) method,

lipids extracted from immature dendritic cells were shown to contain 2-AG, anandamide and the anti-inflammatory anandamide congener, N-palmitoylethanolamine (PalEtn) (2.1 +/- 1.0, 0.14 +/- 0.02 and 8.2 +/- 3.9 pmol x 10(-7) cells, respectively).

………The amounts of 2-AG, but not anandamide or PalEtn, were significantly increased following cell maturation induced by bacterial lipopolysaccharide (LPS) or the allergen Der p 1 (2.8- and 1.9-fold, respectively).

By using both RT-PCR and Western immunoblotting,

………dendritic cells were also found to express measurable amounts of CB1 and CB2 receptors and of FAAH.

Cell maturation did not consistently modify the expression of these proteins, although in some cell preparations a decrease of the levels of both CB1 and CB2 mRNA transcripts was observed after LPS stimulation.

These findings demonstrate for the first time that the endogenous cannabinoid system is present in human dendritic cells and can be regulated by cell activation.


Elphick 2007

Gene. 2007 Sep 1;399(1):65-71. Epub 2007 May 1.


BfCBR: a cannabinoid receptor ortholog in the cephalochordate Branchiostoma floridae (Amphioxus).

Elphick MR.

School of Biological and Chemical Sciences, Queen Mary, University of London, London E1 4NS, UK. M.R.Elphick@qmul.ac.uk

A gene encoding an ortholog of vertebrate CB(1)/CB(2) cannabinoid receptors was recently identified in the urochordate Ciona intestinalis (CiCBR; [Elphick, M.R., Satou, Y., Satoh, N., 2003.

The invertebrate ancestry of endocannabinoid signalling: an orthologue of vertebrate cannabinoid receptors in the urochordate Ciona intestinalis. Gene 302, 95-101.]).

……..Here a cannabinoid receptor ortholog (BfCBR) has been identified in the cephalochordate Branchiostoma floridae. BfCBR is encoded by a single exon and is 410 amino acid residue protein that shares 28% sequence identity with CiCBR and 23% sequence identity with human CB(1) and human CB(2).

The discovery of BfCBR and CiCBR and the absence of cannabinoid receptor orthologs in non-chordate invertebrates indicate that

CB(1)/CB(2)-like cannabinoid receptors originated in an invertebrate chordate ancestor of urochordates, cephalochordates and vertebrates.

Furthermore, analysis of the relationship of BfCBR and CiCBR with vertebrate CB(1) and CB(2) receptors indicates that the gene/genome duplication that gave rise to CB(1) and CB(2) receptors occurred in the vertebrate lineage. Identification of BfCBR, in addition to CiCBR, paves the way for comparative analysis of the expression and functions of these proteins in Branchiostoma and Ciona, respectively,

………..providing an insight into the ancestral functions of cannabinoid receptors in invertebrate chordates prior to the emergence of CB(1) and CB(2) receptors in vertebrates.


Remember, ontogeny recapitulates phylogeny!


Elphick & Egertova 2001

Philos Trans R Soc Lond B Biol Sci. 2001 Mar 29;356(1407):381-408.


The neurobiology and evolution of cannabinoid signalling.

Elphick MR, Egertová M.

School of Biological Sciences, Queen Mary, University of London, London E1 4NS, UK. m.r.elphick@@qmw.ac.uk

The plant Cannabis sativa has been used by humans for thousands of years because of its psychoactivity. The major psychoactive ingredient of cannabis is Delta(9)-tetrahydrocannabinol, which

….exerts effects in the brain by binding to a G-protein-coupled receptor known as the CB1 cannabinoid receptor.

The discovery of this receptor indicated that endogenous cannabinoids may occur in the brain, which act as physiological ligands for CB1.

……….Two putative endocannabinoid ligands,

…arachidonylethanolamide (‘anandamide’) and

….2-arachidonylglycerol, have been identified, giving rise to the concept of a cannabinoid signalling system.

Little is known about how or where these compounds are synthesized in the brain and how this relates to CB1 expression. However, detailed neuroanatomical and electrophysiological analysis of mammalian nervous systems has revealed that the CB1 receptor is targeted to the presynaptic terminals of neurons where it acts to inhibit release of ‘classical’ neurotransmitters.

Moreover, an enzyme that inactivates endocannabinoids, fatty acid amide hydrolase, appears to be preferentially targeted to the somatodendritic compartment of neurons that are postsynaptic to CB1-expressing axon terminals. ………….Based on these findings, we present here a model of cannabinoid signalling in which anandamide is synthesized by postsynaptic cells and acts as a retrograde messenger molecule

…..to modulate neurotransmitter release from presynaptic terminals.

Using this model as a framework, we discuss the role of cannabinoid signalling in different regions of the nervous system in relation to the characteristic physiological actions of cannabinoids in mammals, which include effects on movement, memory, pain and smooth muscle contractility.

The discovery of the cannabinoid signalling system in mammals has prompted investigation of the occurrence of this pathway in non-mammalian animals. Here we review the evidence for the

…….existence of cannabinoid receptors in non-mammalian vertebrates and invertebrates and discuss the

…..evolution of the cannabinoid signalling system.

………Genes encoding orthologues of the mammalian CB1 receptor have been identified in a fish, an amphibian and a bird,

…………indicating that CB1 receptors may occur throughout the vertebrates.

….Pharmacological actions of cannabinoids and specific binding sites for cannabinoids have been reported in several invertebrate species, but the molecular basis for these effects is not known. Importantly, however, the genomes of the protostomian invertebrates Drosophila melanogaster and Caenorhabditis elegans do not contain CB1 orthologues,

………….indicating that CB1-like cannabinoid receptors may have evolved after the divergence of deuterostomes (e.g. vertebrates and echinoderms) and protostomes.

………Phylogenetic analysis of the relationship of vertebrate CB1 receptors with other G-protein-coupled receptors reveals that the paralogues that appear to share the most recent common evolutionary origin with CB1 are lysophospholipid receptors, melanocortin receptors and adenosine receptors.

……..Interestingly, as with CB1, each of these receptor types does not appear to have Drosophila orthologues, indicating that this group of receptors may not occur in protostomian invertebrates.

We conclude that the cannabinoid signalling system may be quite restricted in its phylogenetic distribution, probably occurring only in the deuterostomian clade of the animal kingdom and possibly only in vertebrates.


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