In 2007, AstraZeneca researchers showed that an orphan receptor called GPR55 is activated by THC, anandamide, and other cannabinoids — raising the question of whether the endocannabinoid system has a third receptor. Nearly two decades later, the debate is still unresolved.
Anyone interested in the boundaries of the endocannabinoid system, how CBD might work beyond CB1/CB2, or why cannabinoid pharmacology is more complex than "two receptors, two endocannabinoids."
13.5%amino acid sequence homology between GPR55 and CB1 — barely related at the genetic level, yet activated by the same cannabinoid ligands. This paradox is why the debate about whether GPR55 is truly "CB3" remains unresolved after nearly two decades.
The Backstory
By 2007, the endocannabinoid system had a tidy architecture: two receptors (CB1 in the brain, CB2 in immune cells), two endocannabinoids (anandamide, 2-AG), and the enzymes that make and degrade them. Textbooks were being written. The system felt complete.
But cannabinoid effects kept appearing that neither CB1 nor CB2 could explain. Vascular responses. Bone remodeling. Pain phenomena in knockout mice that shouldn't have happened if only two receptors existed. Something else was responding to cannabinoids. Something nobody had identified.
At a pharmaceutical company in Sweden, researchers testing orphan receptors were about to find it — and spark a debate that still hasn't been settled.
An Orphan Gets a Name
GPR55 was one of thousands of orphan G-protein-coupled receptors — genes encoding receptor proteins whose function and natural ligands were unknown. AstraZeneca's drug discovery division in Mölndal, Sweden, was systematically testing these orphans against panels of known drug molecules to figure out what they respond to. It's a pharmaceutical fishing expedition: transfect cells with the orphan receptor gene, expose them to hundreds of compounds, and see what activates them.
When they tested GPR55 against cannabinoids, the results were striking.
How They Did It
How AstraZeneca Identified GPR55 as Cannabinoid-Responsive
Transfect cells
HEK293 cells were engineered to express human GPR55 on their surface — cells that don't normally have this receptor were given the gene.
Standard approach for studying orphan receptors in isolation
Test cannabinoid panel
The transfected cells were exposed to a panel of cannabinoid ligands: CP55940, THC, anandamide, 2-AG, virodhamine, noladin ether, and CBD among others.
Both plant-derived and endogenous cannabinoids tested
Measure activation
GTPγS binding assays measured whether each compound activated GPR55. THC activated it with an EC50 of 8 nanomolar — remarkably potent. Endocannabinoids activated it at similar nanomolar concentrations.
8 nM is more potent than THC at CB1 in some assay conditions
Identify G-protein coupling
Using antibody and peptide blocking approaches, the team determined that GPR55 couples to Gα13 — NOT the Gi/o proteins used by CB1 and CB2. This means completely different downstream signaling.
Gα13 activates rhoA, cdc42, rac1 — cytoskeletal regulation, not adenylate cyclase
Test CBD
CBD did not activate GPR55 but antagonized it — blocking the effect of CP55940 with an IC50 of 445 nM. CBD is a GPR55 blocker, not an activator.
This has implications for CBD's anti-cancer mechanism
Ryberg et al. (2007), Br J Pharmacol 152:1092-1101
The headline finding: GPR55 responds to the same cannabinoid molecules that activate CB1 and CB2. THC, anandamide, 2-AG — they all bind and activate this orphan receptor. If it looks like a cannabinoid receptor and it responds like a cannabinoid receptor...
The paper's title made the claim directly: "The orphan receptor GPR55 is a novel cannabinoid receptor."
The field immediately pushed back.
The Case for CB3
Evidence FOR (CB3)
moderate- Activated by THC at 8 nM — potent, specific response
- Activated by endocannabinoids (anandamide, 2-AG, virodhamine, noladin ether) at nanomolar concentrations
- CBD antagonizes it — consistent with CBD's multi-target pharmacology
- GPR55 knockout mice show altered pain, bone density, and cancer phenotypes — physiologically relevant
- Expressed in brain, GI tract, bone, immune cells — overlapping with ECS distribution
Evidence AGAINST (Not CB3)
strong- Only 13.5% sequence homology with CB1 and 14.4% with CB2 — barely related genetically
- Couples to Gα13, not Gi/o — completely different intracellular signaling cascade
- Primary endogenous ligand is lysophosphatidylinositol (LPI), a non-cannabinoid lipid (Oka et al., 2007)
- 2024 crystal structure shows binding pocket is entirely different from CB1/CB2
- IUPHAR has NOT officially designated it as a cannabinoid receptor
- Many cannabinoid effects at GPR55 only seen at overexpression — may not occur at native levels
Current Consensus
moderate- GPR55 is a 'putative' or 'atypical' cannabinoid receptor
- It clearly responds to some cannabinoids but its pharmacology is fundamentally different
- Whether it belongs in the cannabinoid receptor family is partly a naming question
- Its biology matters regardless of what we call it
After nearly two decades of debate, the field has landed on a pragmatic middle ground: GPR55 responds to cannabinoids and has physiological relevance, but it's so pharmacologically different from CB1 and CB2 that calling it 'CB3' implies a kinship that may not exist at the molecular level. The biology matters more than the label.
Ryberg et al. (2007); Oka et al. (2007); Sharir & Bhanu (2010), PMC2874616; Cell Research (2024)
The same year Ryberg published this paper, Shingo Oka's group in Japan demonstrated that GPR55's endogenous ligand is lysophosphatidylinositol (LPI) — a lipid that is not an endocannabinoid. If the receptor's natural activator isn't a cannabinoid, can the receptor truly be called a cannabinoid receptor?
Roger Pertwee wrote an accompanying editorial in the same journal issue titled "GPR55: a new member of the cannabinoid receptor clan?" — the question mark in the title telling you everything about the field's uncertainty.
How Different Is GPR55, Really?
GPR55 (The Candidate)
- ~350 amino acids
- 13.5% homology with CB1, 14.4% with CB2
- Couples to Gα13 → rhoA/rac1/cdc42
- Endogenous ligand: LPI (non-cannabinoid)
- Also activated by THC, anandamide, 2-AG
- CBD is an antagonist (IC50 = 445 nM)
- Found in brain, bone, gut, immune cells
Cannabinoid-responsive, but fundamentally different machinery
CB1 (The Brain Receptor)
- 472 amino acids
- 97-99% conserved across mammals
- Couples to Gi/o → adenylate cyclase inhibition
- Endogenous ligands: anandamide, 2-AG
- THC is a partial agonist
- CBD is a negative allosteric modulator
- Most abundant GPCR in the brain
Classical cannabinoid receptor — established, defined, central
CB2 (The Immune Receptor)
- 360 amino acids
- 44% homology with CB1
- Couples to Gi/o → adenylate cyclase inhibition
- Endogenous ligands: anandamide, 2-AG
- Non-psychoactive when activated
- Primarily in immune cells and activated microglia
- CB2-selective agonists in clinical development
Classical cannabinoid receptor — immune-focused, non-psychoactive
Ryberg et al. (2007); Matsuda et al. (1990); Munro et al. (1993); Sharir & Bhanu (2010)
The comparison makes the problem clear. CB1 and CB2 share 44% of their sequence, use the same G-protein family (Gi/o), respond to the same endocannabinoids, and inhibit the same enzyme (adenylate cyclase). They're recognizably related — siblings with different jobs.
GPR55 shares only 13-14% sequence identity with either. It uses a completely different G-protein (Gα13). Its natural ligand isn't a cannabinoid. Its downstream signaling (cytoskeletal regulation via rhoA) has nothing in common with classical cannabinoid signaling (adenylate cyclase inhibition). It responds to cannabinoids, but it's built differently, signals differently, and evolved for something different.
Is a receptor that responds to cannabinoids automatically a cannabinoid receptor? Or is "cannabinoid receptor" a functional family defined by more than just ligand overlap?
The field hasn't agreed.
What GPR55 Actually Does
Regardless of whether it's called CB3, GPR55 has real biology that matters:
Results
GPR55 Knockout Phenotypes — What Happens Without It
GPR55 knockout mouse studies; Whyte et al. (2009); Staton et al. (2008)
The bone finding is particularly intriguing. GPR55 promotes osteoclast function — the cells that break down bone. Without GPR55, mice have denser, stronger bones. This suggests GPR55 antagonists could treat osteoporosis — a therapeutic angle completely unrelated to cannabis but enabled by this receptor's cannabinoid responsiveness.
The cancer connection is equally compelling. GPR55 is overexpressed in multiple cancer types — acute myeloid leukemia, uveal melanoma, glioma, renal cancer. Tumor cells appear to co-opt GPR55 signaling to increase motility and aggressiveness. Blocking GPR55 reduces cancer cell migration.
Which brings us to CBD.
Why CBD Blocks GPR55 — and Why That Matters
One of the paper's most practically important findings was that CBD is a potent GPR55 antagonist — it blocks the receptor at nanomolar concentrations (IC50 = 445 nM) without activating it.
This has direct implications for understanding CBD's therapeutic profile:
Biological Mechanism
CBD → GPR55 Antagonism → Potential Anti-Cancer Effects
GPR55 overexpressed in tumors
Multiple cancer types show elevated GPR55 expression, which promotes cell migration and aggressiveness.
LPI activates GPR55 on tumor cells
Tumor cells produce lysophosphatidylinositol, which activates GPR55, driving downstream signaling through Gα13/rhoA that reorganizes the cytoskeleton for cell movement.
CBD blocks GPR55
CBD antagonizes GPR55 at nanomolar potency (IC50 = 445 nM), preventing LPI-driven activation and reducing the signaling that drives migration.
Reduced tumor aggressiveness
GPR55 blockade reduces cancer cell motility and potentially limits metastatic spread. This represents one proposed mechanism for CBD's anti-cancer effects observed in preclinical models.
Ryberg et al. (2007); Ford et al. (2010); Andradas et al. (2022)
This doesn't prove CBD cures cancer — the evidence is preclinical and far from clinical application. But it does provide a mechanistic pathway through which CBD's anti-cancer effects in laboratory models might work, independently of cannabinoid receptors CB1 and CB2.
The Bigger Question: How Many Receptors Are There?
Myth vs. Reality
The endocannabinoid system has two receptors — CB1 and CB2. That's it.
Multiple other receptors respond to cannabinoids. GPR55, GPR18, and GPR119 are all G-protein-coupled receptors activated by various cannabinoids. TRPV1 (a vanilloid receptor) is activated by anandamide and CBD. PPARα and PPARγ (nuclear receptors) respond to both endocannabinoids and phytocannabinoids. The 'two receptors, two endocannabinoids' model is a useful simplification, not the full picture.
The Evidence
Ryberg et al. (2007) demonstrated GPR55 responds to THC and endocannabinoids. Anandamide was shown to activate TRPV1 by Zygmunt et al. (1999). CBD activates 5-HT1A serotonin receptors. The endocannabinoid system's boundaries are blurrier than textbooks suggest — it overlaps with the endovanilloid system, serotonin signaling, and lipid signaling networks that extend far beyond two receptors.
Ryberg et al. (2007); Zygmunt et al. (1999); Pertwee (2008)
GPR55's story is really about the limits of tidy categories. The endocannabinoid system was discovered through cannabis — we found the receptors by looking for what THC binds. But the body's lipid signaling networks don't respect the boundaries we drew around "the cannabinoid system." GPR55 may be part of it, or it may be part of a different but overlapping system. The molecules don't care what we call the receptor families. They bind what they bind.
This is why cannabinoid pharmacology is more complex than most popular descriptions suggest — and why cannabis produces such a wide range of effects. The plant's molecules interact with a network of receptors that extends beyond the two we've named "cannabinoid."
Related Research
The Receptors — Known and Debated
GPR55 challenges the assumption that the endocannabinoid system has only two receptors. These are the studies that established what we know — and what we're still figuring out.
Structure of a cannabinoid receptor and functional expression of the cloned cDNA
Matsuda et al. (1990)
CB1 — the established brain receptor GPR55 is compared to (13.5% homology)
Molecular characterization of a peripheral receptor for cannabinoids
Munro et al. (1993)
CB2 — the established immune receptor (14.4% homology with GPR55)
The diverse CB1 and CB2 receptor pharmacology of three plant cannabinoids
Pertwee (2008)
Characterized CBD as a multi-target antagonist — including GPR55 blockade
Isolation, Structure, and Partial Synthesis of an Active Constituent of Hashish
Gaoni & Mechoulam (1964)
THC — the molecule that activates GPR55 at 8 nM, even more potently than at some CB1 assay conditions
Is GPR55 the third cannabinoid receptor (CB3)?
It's genuinely unclear — and that's what makes this study important. GPR55 is activated by THC, anandamide, 2-AG, and other cannabinoids, which makes it look like a cannabinoid receptor. But it shares only 13-14% of its amino acid sequence with CB1 or CB2, uses completely different intracellular signaling (Gα13 instead of Gi/o), and its primary natural ligand is a non-cannabinoid lipid called lysophosphatidylinositol (LPI). The International Union of Pharmacology (IUPHAR) has not officially designated it as CB3. Most researchers describe it as a "putative" or "atypical" cannabinoid receptor. The biology matters more than the label.
Why does it matter whether GPR55 is a cannabinoid receptor or not?
Because it affects how we understand both the scope of the endocannabinoid system and how CBD works. CBD is a potent GPR55 antagonist (blocker), and GPR55 is overexpressed in several cancer types where it promotes tumor cell migration. CBD's anti-cancer effects in laboratory models may partly work through GPR55 blockade — a mechanism completely independent of CB1 or CB2. Whether we call it "CB3" or not, GPR55 clearly responds to cannabinoids and has real physiological relevance for bone density, pain, and cancer.
What the researchers found
GPR55 binds to and is activated by the cannabinoid ligand CP55940 and by THC (EC50 = 8 nM, remarkably potent). Endocannabinoids including anandamide, 2-AG, virodhamine, and noladin ether activate GPR55 with nanomolar potencies. CBD does not activate GPR55 but antagonizes the agonist effect of CP55940 (IC50 = 445 nM). GPR55 couples to Gα13 protein (not Gi/o like CB1 and CB2) and activates downstream rhoA, cdc42, and rac1 signaling — a cytoskeletal regulation pathway entirely different from the adenylate cyclase inhibition of classical cannabinoid receptors.
Why it matters
This paper challenged the assumption that the endocannabinoid system has only two receptors. If GPR55 is a genuine cannabinoid receptor, the ECS is larger and more complex than previously understood. More practically, GPR55 may explain cannabinoid effects that CB1 and CB2 cannot account for, and CBD's antagonism of GPR55 may contribute to its anti-cancer and anti-inflammatory effects. The paper also raised a broader question: how many receptors does the endocannabinoid system actually have?
How the study worked
HEK293 cells were transiently transfected with human GPR55 cDNA. Radioligand binding assays determined ligand affinity. GTPγS binding assays measured receptor activation by a panel of cannabinoid and non-cannabinoid ligands at various concentrations. G-protein coupling was determined using antibody and peptide blocking approaches targeting specific Gα subunits. Downstream signaling was assessed by measuring activation of rhoA, cdc42, and rac1 GTPases. Controls included untransfected cells and cells expressing CB1 or CB2 for comparison.
What this study cannot tell us
This is an in vitro study using transfected cell lines — GPR55 was overexpressed far beyond physiological levels, which may produce pharmacological effects not seen at native expression. The paper came from AstraZeneca's drug discovery division (industry, not academic). The cannabinoid specificity of GPR55 was challenged the same year by Oka et al., who showed that the non-cannabinoid lipid lysophosphatidylinositol (LPI) robustly activates GPR55. The low sequence homology (13-14%) with CB1/CB2 and the different G-protein coupling (Gα13 vs Gi/o) raise fundamental questions about whether GPR55 belongs in the cannabinoid receptor family at all.
How to read the evidence
Rated moderate because while the in vitro pharmacological evidence for cannabinoid activation of GPR55 is robust, the receptor's status as a true cannabinoid receptor remains debated. The low sequence homology with CB1/CB2, different G-protein coupling, and the identification of a non-cannabinoid endogenous ligand (LPI) complicate the picture. IUPHAR has not officially designated GPR55 as a cannabinoid receptor.
When this study was published
Published in 2007, this 19-year-old paper launched a debate that continues today. The core finding — that GPR55 responds to cannabinoid ligands — has been replicated. But the interpretation — that GPR55 is a cannabinoid receptor — remains contested. A 2024 crystal structure of GPR55 showed its binding pocket is entirely different from CB1/CB2.
The bigger picture
GPR55 sits at the intersection of two unresolved questions: (1) How many receptors does the endocannabinoid system actually have? Beyond CB1 and CB2, GPR55, GPR18, GPR119, and TRPV1 all respond to cannabinoids to varying degrees. (2) How does CBD work? CBD antagonizes GPR55 at nanomolar potency — this may contribute to CBD's anti-cancer effects (GPR55 promotes cancer cell migration and is overexpressed in several tumors) and anti-inflammatory effects. The pharmaceutical industry recognized GPR55 as a potential drug target: GPR55 knockout mice show altered bone density, pain sensitivity, and cancer susceptibility, suggesting genuine therapeutic relevance regardless of the naming debate.
Questions still open
- Is GPR55 a true cannabinoid receptor (CB3) or just a receptor that happens to respond to some cannabinoids?
- Does CBD's antagonism of GPR55 explain some of its anti-cancer and anti-inflammatory effects?
- How many receptors does the endocannabinoid system actually encompass?
Common questions
Is GPR55 the third cannabinoid receptor (CB3)?
Why does it matter whether GPR55 is a cannabinoid receptor or not?
Read the original research
The orphan receptor GPR55 is a novel cannabinoid receptor
British Journal of Pharmacology, 152(7), 1092-1101
Citation
Ryberg, E; Larsson, N; Sjögren, S; Hjorth, S; Hermansson, N O; Leonova, J; Elebring, T; Nilsson, K; Drmota, T; Greasley, P J. (2007). The orphan receptor GPR55 is a novel cannabinoid receptor. British Journal of Pharmacology, 152(7), 1092-1101. https://doi.org/10.1038/sj.bjp.0707460