In 1993, a Cambridge laboratory cloned a second cannabinoid receptor found not in the brain but in immune cells — revealing that cannabinoids modulate the immune system through a dedicated molecular target.
Anyone interested in how cannabis affects the immune system, why cannabinoids reduce inflammation, or the search for anti-inflammatory cannabis-based medicines without psychoactive effects.
44%amino acid identity with CB1 — making CB2 a distant cousin, not a twin. Different enough to have entirely different tissue distribution, different functions, and different therapeutic potential
The Backstory
By 1993, the endocannabinoid system was taking shape. THC had been identified. The brain's cannabinoid receptor — CB1 — had been cloned. Anandamide had been found. But there was a problem. Cannabis doesn't just affect the brain. It suppresses inflammation, alters immune cell function, reduces swelling, modulates antibody production. CB1 is overwhelmingly a brain receptor. Something else had to be mediating the immune effects.
At the MRC Laboratory of Molecular Biology in Cambridge — the lab where DNA's structure was determined, where monoclonal antibodies were invented, the most decorated molecular biology laboratory in history — a team was about to find it hiding in cancer cells.
Cloning from Cancer Cells
Sean Munro was not a cannabinoid researcher. He was a cell biologist whose primary interest was the Golgi apparatus — the cellular machinery that processes and sorts proteins. He had been a group leader at the MRC-LMB since 1989. But in the early 1990s, the rush to clone G-protein-coupled receptors was drawing labs from across molecular biology into receptor hunting, and Munro's team had the tools.
They started with HL-60 cells — a human promyelocytic leukemia cell line widely used in immunology research. These cells can be chemically differentiated into granulocyte-like immune cells, making them a rich source of immune-relevant receptor genes.
Process
How CB2 Was Found
Differentiate HL-60 cells
Human leukemia cells were treated with dimethylformamide to push them toward granulocyte (immune cell) identity, activating immune-relevant gene expression.
Hunt for unknown GPCRs
PCR with degenerate primers — short DNA sequences matching conserved regions across the GPCR family — was used to fish for novel receptor genes in the cDNA library.
Find a CB1 relative
One clone showed clear homology to the G-protein-coupled receptor family and partial sequence similarity to CB1 — but only 44% amino acid identity. A distant cousin, not a twin.
Express and test
The full gene was cloned, inserted into cultured cells, and tested against cannabinoid compounds. It inhibited adenylate cyclase via Gi/o proteins — confirming cannabinoid receptor pharmacology.
Map the distribution
In situ hybridization and Northern blots revealed high CB2 mRNA in the spleen — specifically in marginal zone macrophages. The receptor was undetectable in brain, liver, thymus, lung, and kidney.
Munro et al. (1993), Nature 365:61-65
The tissue distribution was the surprise. This wasn't another brain receptor. CB2 was in the immune system — spleen, tonsils, white blood cells. Cell sorting pinpointed the signal to monocytes and macrophages. The paper called it "a peripheral receptor for cannabinoids," and the label stuck for decades.
360 Amino Acids, 44% Identity
44%
amino acid identity between CB2 and CB1 — making them distant cousins within the GPCR superfamily. For comparison, the human and rat versions of CB1 share 97-99% identity. CB2's low homology with CB1 means it has a different binding pocket, different signaling properties, and different pharmacological potential.
Munro et al. (1993), Nature; Zou & Kumar (2018), PMC5877694
The paper was published September 2, 1993, in Nature — the same journal that had published CB1's cloning three years earlier. Five pages. Three authors from one of the most storied laboratories in science.
What Munro, Thomas, and Abu-Shaar had found:
- A G-protein-coupled receptor, 360 amino acids, seven transmembrane domains
- Gi/o-coupled, inhibits adenylate cyclase — same signaling family as CB1
- Only 44% amino acid identity with CB1 (cross-species CB2 homology is ~80%, compared to CB1's 97-99%)
- Two human isoforms: one predominantly in testis, the other mainly in spleen
- Gene designated CNR2, GenBank accession X74328
The endocannabinoid system now had two receptors. But they were not equals. CB1 got the attention — it was in the brain, it mediated the high, it was the target for drug development. CB2 was "peripheral." Immune. Unglamorous.
That would change.
The Sibling Receptors
CB2 (This Paper)
- 360 amino acids
- Found primarily in immune cells (spleen, macrophages, white blood cells)
- Originally reported absent from the brain
- Activation is non-psychoactive — no high
- Primary role: immune regulation, inflammation
- Gene: CNR2, cloned 1993 from HL-60 cells
- Only 44% identity with CB1
The immune receptor — non-psychoactive, anti-inflammatory
CB1 (Matsuda, 1990)
- 472 amino acids
- Most abundant GPCR in the mammalian brain
- Dense in hippocampus, cerebellum, basal ganglia, cortex
- Activation produces the psychoactive effects of THC
- Primary role: neuromodulation, synaptic signaling
- Gene: CNR1, cloned 1990 from rat brain
- 97-99% conserved across mammals
The brain receptor — psychoactive, abundant, complex
Munro et al. (1993), Nature; Matsuda et al. (1990), Nature; Zou & Kumar (2018)
Both receptors are GPCRs. Both couple to Gi/o proteins. Both bind THC and the endocannabinoids anandamide and 2-AG. But their distribution and function are fundamentally different — which is exactly what makes CB2 therapeutically interesting. If you could activate CB2 without touching CB1, you might get the anti-inflammatory and analgesic effects of cannabinoids without the psychoactive effects. Pain relief without the high.
That idea has driven two decades of drug development. The results have been... complicated.
The Identity Crisis
The 1993 characterization was clean: CB2 in immune cells, not in brain. But science rarely stays that simple.
The Original View (1993-2005)
strong- Munro's paper found no CB2 mRNA in brain tissue
- Multiple groups confirmed: Northern blots, RT-PCR, and radioligand binding showed no CB2 in cortex, cerebellum, or whole brain
- CB2 became 'the peripheral cannabinoid receptor' — a label that persisted in textbooks for over a decade
The Challenge (2005-present)
moderate- CB2 mRNA and protein found on activated microglia (brain immune cells) during neuroinflammation — absent in healthy brain, upregulated in disease
- Van Sickle (2005): CB2 in dorsal motor nucleus of vagus nerve, colocalized with neuronal markers, not glial markers
- Some groups report CB2 in hippocampal neurons, brainstem, cortex, dorsal root ganglia
- But antibody specificity is a major concern — some 'positive' results don't replicate in CB2 knockout tissue
Current Consensus
strong- CB2 is definitively expressed in immune cells and activated brain microglia
- CB2 is probably expressed in some neurons, at least under pathological conditions
- CB2 is NOT abundant in healthy brain neurons the way CB1 is
- The 'peripheral only' label was wrong, but CB2 remains primarily an immune receptor
The debate was partly methodological — early tools weren't sensitive enough to detect low-level expression, but later tools were sometimes too sensitive, amplifying signals from contaminating immune cells in brain tissue. The current view is nuanced: CB2 is not a brain receptor in the way CB1 is, but it is not absent from the brain either. Its role there appears to be primarily inflammatory and protective rather than psychoactive.
Atwood & Mackie (2010), PMC2931549; Zou & Kumar (2018), PMC5877694
The "identity crisis" — as a landmark 2010 review called it — matters because it determines CB2's therapeutic scope. If CB2 is only on immune cells, it's a target for peripheral inflammation and pain. If it's also on brain cells during disease states, it could be a target for Alzheimer's, Parkinson's, stroke, and traumatic brain injury — conditions where neuroinflammation drives damage.
Not Just Peripheral
The brain mapping that has emerged since 1993 tells a more complex story than the original paper suggested.
Results
CB2 Expression Across Tissues and Conditions
Atwood & Mackie (2010); Zou & Kumar (2018); Turcotte et al. (2016)
The critical insight: CB2 is an inducible receptor in the brain. In a healthy brain, it's essentially absent. During neuroinflammation — a stroke, a traumatic injury, Alzheimer's progression — microglia activate and CB2 expression surges. This makes it a potential therapeutic target specifically for disease states, not for baseline brain function. An elegant biological design: a receptor that appears when and where it's needed.
The Most Promising Target That Hasn't Worked Yet
The therapeutic logic for CB2 drugs is irresistible: activate CB2 to reduce pain and inflammation without any psychoactive effects. No high, no cognitive impairment, no abuse potential. Decades of preclinical research have validated this concept — in animals.
Research Timeline
CB2: From Discovery to Clinical Disappointment
CB2 cloned from immune cells (Munro, Cambridge)
Second cannabinoid receptor identified — peripheral, non-psychoactive
SR144528: first selective CB2 antagonist developed
Tool compound enables CB2-specific pharmacology research
CB2 found in brain neurons for the first time (Van Sickle)
Challenges the 'peripheral only' dogma
CB2 agonists shown to be neuroprotective in Alzheimer's model
CB2 activation reduces neuroinflammation and neuronal death
'CB2: a cannabinoid receptor with an identity crisis' published
Landmark review documenting the brain expression debate
CB2 agonist LY2828360 enters Phase 2 trial for osteoarthritis
The most advanced CB2 drug candidate reaches human testing
LY2828360 fails Phase 2 — no significant pain reduction
The leading CB2 drug candidate doesn't work in humans
No CB2-selective drug has been approved for any indication
Despite 30 years of research and stunning preclinical data
Atwood & Mackie (2010); Frontiers in Pharmacology (2022), PMC9585503
The preclinical results are genuinely impressive. CB2 agonists reduce inflammation in arthritis models, provide analgesia in neuropathic pain models, show neuroprotection in Alzheimer's and stroke models, reduce fibrosis in liver disease, and slow tumor growth in some cancer models. The data in animals is as good as any target in pharmacology.
But none of it has translated to humans.
The failures likely stem from a convergence of problems: species differences in CB2 expression patterns between rodents and humans, the difficulty of reliably detecting CB2 protein in human tissues (antibody specificity remains a major challenge), and the possibility that CB2's role in human physiology differs from its role in mice in ways we don't yet understand.
Where CB2 Research Stands Now
CB2 remains an active area of research — arguably more active now than at any point since its discovery. Current work focuses on:
- Neuroinflammation: CB2 activation on microglia as a target for neurodegenerative diseases
- Better tools: Developing reliable antibodies and imaging agents for CB2 detection in human tissue
- Allosteric modulators: Drugs that fine-tune CB2 rather than fully activating it
- Peripherally-restricted agonists: Ensuring CB2 drugs stay out of the brain (ironic, given that brain CB2 might be therapeutically useful)
- Combination approaches: Using CB2 activation alongside other anti-inflammatory strategies
The endocannabinoid system that CB2 helped define is now recognized as one of the most important regulatory networks in human physiology. CB2's specific contribution — the immune and inflammatory arm of that system — may yet yield the therapeutic breakthroughs its discovery promised. But the gap between preclinical promise and clinical reality remains one of the defining frustrations of cannabinoid pharmacology.
Myth vs. Reality
CB2 is only found in the immune system and has nothing to do with the brain.
CB2 was originally described as a 'peripheral receptor' absent from the brain. Three decades of subsequent research have shown that CB2 is expressed in brain microglia during neuroinflammation and possibly in some neurons. It's not a brain receptor the way CB1 is, but it's not absent from the brain either.
The Evidence
Van Sickle et al. (2005) demonstrated CB2 in brainstem neurons. Multiple groups have shown CB2 upregulation on activated microglia in Alzheimer's, stroke, and TBI models. The 'identity crisis' review (Atwood & Mackie, 2010) documents the evolving understanding. However, some positive findings have failed to replicate in CB2 knockout control tissue, so the exact extent of neuronal expression remains debated.
Atwood & Mackie (2010), PMC2931549; Van Sickle et al. (2005), Nature
Related Research
The Receptor Family
CB2 is the second of two known cannabinoid receptors. Its discovery completed the receptor foundation of the endocannabinoid system — though there may yet be more to find.
Structure of a cannabinoid receptor and functional expression of the cloned cDNA
Matsuda et al. (1990)
The sibling that got all the attention — CB1, the brain's cannabinoid receptor
Isolation and structure of a brain constituent that binds to the cannabinoid receptor
Devane, Hanuš, Mechoulam et al. (1992)
The first endocannabinoid — binds both CB1 and CB2, but with different affinity and efficacy
GPR55 as novel cannabinoid receptor
Ryberg et al. (2007)
The possible 'CB3' — another GPCR that responds to some cannabinoids, raising the question of how many cannabinoid receptors exist
Isolation, Structure, and Partial Synthesis of an Active Constituent of Hashish
Gaoni & Mechoulam (1964)
The molecule that started it all — THC binds both CB1 and CB2
What is the CB2 receptor and how is it different from CB1?
CB2 is the second cannabinoid receptor, found primarily in immune cells (spleen, white blood cells, macrophages) rather than in the brain. It shares only 44% of its amino acid sequence with CB1. While CB1 mediates the psychoactive effects of THC — the high — CB2 activation is non-psychoactive and primarily modulates immune and inflammatory responses. Both receptors are part of the endocannabinoid system and both respond to THC and the body's own endocannabinoids, but they serve fundamentally different physiological roles.
Could CB2 drugs treat pain without getting you high?
That's been the hope for thirty years. Because CB2 is primarily on immune cells rather than brain neurons, drugs that selectively activate CB2 should reduce inflammation and pain without psychoactive effects. Preclinical studies in animals have been very promising — CB2 agonists work in models of arthritis, neuropathic pain, and neuroinflammation. However, no CB2-targeted drug has succeeded in human clinical trials to date. Species differences in CB2 expression, difficulty detecting the receptor reliably in human tissues, and poor translation from animal models to human biology are the likely reasons.
What the researchers found
A gene encoding a novel G-protein-coupled receptor was cloned from the human promyelocytic leukemia cell line HL-60. The receptor shared only 44% amino acid sequence identity with the previously cloned CB1 receptor. When expressed in cells, it exhibited cannabinoid binding properties and inhibited adenylate cyclase via Gi/o proteins. In situ hybridization and Northern blot analysis revealed high CB2 mRNA levels in spleen — specifically in marginal zone macrophages — but not in brain, liver, thymus, lung, or kidney. The receptor was designated CB2 and termed the 'peripheral cannabinoid receptor.' The protein consists of 360 amino acids with the seven-transmembrane topology characteristic of GPCRs.
Why it matters
This paper established that the endocannabinoid system extends beyond the brain into the immune system. CB2 explained a longstanding observation: that cannabis suppresses inflammation and modulates immune responses. Because CB2 activation doesn't produce psychoactive effects, it became the most attractive therapeutic target in cannabinoid pharmacology — a way to harness cannabis's anti-inflammatory and analgesic properties without the high. The discovery also expanded the endocannabinoid system from a brain signaling network to a body-wide regulatory system.
How the study worked
Human promyelocytic leukemia cells (HL-60) were treated with dimethylformamide to induce granulocyte differentiation. A cDNA library was prepared and screened using PCR with degenerate primers targeting conserved G-protein-coupled receptor transmembrane domains. One clone showed homology to the GPCR family and partial sequence identity with CB1. The full-length cDNA was cloned, sequenced, and expressed in cultured cells to confirm cannabinoid receptor pharmacology. A rat homologue was also isolated by PCR. Tissue distribution was mapped using in situ hybridization, Northern blotting, and RT-PCR across multiple rat tissues. Cell sorting of spleen populations identified the macrophage/monocyte population as the primary CB2-expressing cells.
What this study cannot tell us
This was a molecular cloning and tissue distribution study performed in cell lines and rodent tissues, not in intact humans. The initial characterization that CB2 was absent from the brain was based on the sensitivity limits of 1993 techniques — later studies with more sensitive methods detected CB2 in brain microglia and possibly neurons. The functional assays were performed in transfected cell lines, not in native immune cells. Species differences in CB2 expression patterns (particularly between rodents and humans) later proved to be a major obstacle for translating preclinical findings to clinical use.
How to read the evidence
Rated strong because this is a definitive molecular cloning result confirmed by multiple independent groups. The CB2 gene (CNR2) has been sequenced across species, the receptor's pharmacology is well-characterized, and its role in immune function has been validated by extensive subsequent research including knockout mouse studies.
When this study was published
Published in 1993, this 33-year-old paper established the molecular identity of the second cannabinoid receptor. The core finding — that CB2 is expressed in immune cells and responds to cannabinoids — is firmly established. The original claim that CB2 is absent from the brain has been revised; CB2 is now known to be expressed in activated microglia and possibly some neurons.
The bigger picture
CB2 completed the receptor foundation of the endocannabinoid system: two receptors (CB1 in brain, CB2 in immune cells), two endogenous ligands (anandamide and 2-AG), and the enzymes that make and degrade them. But CB2's story didn't end with its discovery. The original characterization as 'peripheral only' was progressively challenged — CB2 was found on activated microglia during neuroinflammation and possibly on some neurons. This 'identity crisis' transformed CB2 from an immune receptor to a potential neurotherapeutic target for Alzheimer's, stroke, traumatic brain injury, and neuroinflammatory conditions. Despite stunning preclinical results, every CB2 drug clinical trial has failed to date, making it one of the great unfulfilled promises in cannabinoid medicine.
Questions still open
- If CB2 is on immune cells, could targeting it treat autoimmune and inflammatory diseases without immunosuppressive side effects?
- Is CB2 truly absent from the brain, or was it just below the detection limit of 1993 methods?
- Can CB2 agonists provide pain relief without the psychoactive effects of CB1 activation?
Common questions
What is the CB2 receptor and how is it different from CB1?
Could CB2 drugs treat pain without getting you high?
Read the original research
Molecular characterization of a peripheral receptor for cannabinoids
Nature, 365(6441), 61-65
Citation
Munro, S; Thomas, K L; Abu-Shaar, M. (1993). Molecular characterization of a peripheral receptor for cannabinoids. Nature, 365(6441), 61-65. https://doi.org/10.1038/365061a0