CB1 and CB2 receptors are the members of class A GPCR family, so they share a common structural feature of this class, having an extracellular glycosylated amino N-terminal and an intracellular carboxy C-terminal. The terminals are connected by seven transmembrane (TM) regions, three intracellular (ICL1, ICL2, ICL3) and three extracellular (ECL1, ECL2, ECL3) loops and an amphipathic helix 8. CB2 receptor shows 44% sequence similarity and high degree of homology with CB1. There are 2 highly conserved regions, NPxxY motif and DRY motif, whose residues adopt almost the same position in all the active structures present till now. CB1 and CB2 receptors are coupled to G-proteins,which comprise of α, β, γ subunits. Therefore, they participate in G-protein (Gi/o family) mediated signaling pathways. Structurally different ligands when bind to the receptor can activate different signal transduction pathways, which in turn mediates various downstream signaling cascades. This process termed as “biased signaling” or functional selectivity, can provide therapeutical advantage by targeting a specific pathway to get desired effect. Cannabinoid receptors (CB1 and CB2), as part of the endocannabinoid system, play a critical role in various physiological and pathological conditions. Thus, many efforts are been made to develop ligands for CB1 and CB2 receptors, resulting in number of phyto and synthetic cannabinoids with varying affinities for the treatment of various diseases.

Mutations in Cannabinoid receptor 1
Residue Position Transmembrane Region Effect References
F238L TM4 Enhances Basal Endocytosis via Lipid Rafts https://doi.org/10.1016%2Fj.brainres.2006.05.042
L207A TM3 Higher constitutive activity, demonstrated by a higher basal-specific GTPγS binding https://doi.org/10.1016%2Fj.brainres.2006.05.042
T210I TM3 Mutations of CB1 T210 produce active and inactive receptor forms: correlations with ligand affinity, receptor stability, and cellular localization https://doi.org/10.1016%2Fj.brainres.2006.05.042
F201A TM3 Increased basal [35S]GTPγS binding of the receptor https://doi.org/10.3390/ijms20081837
T210A TM3 Lower constitutive activity and had an increased thermal stability, suggestive of a shift towards the inactive state https://doi.org/10.3390/ijms20081837
R214A TM3 Partial decrease in its ability to activate heterotrimeric Go proteins https://doi.org/10.1530/JME-14-0219
Y215A TM3 Increases constitutive activity of CB1R https://doi.org/10.1530/JME-14-0219
F237L TM4 Hindered Outward Movement of TM6 https://doi.org/10.1021/acsomega.2c04980
C415A C-Ter Loss of palmitoylation, marked loss of association with lipid rafts on the plasma membrane and loss of activity, when assayed for downstream GTP-binding and reduction in cAMP levels https://doi.org/10.1111/j.1476-5381.2011.01658.x
N393A TM7 Impair β-arrestin signaling, with no effect on G-protein signaling https://doi.org/10.1089/can.2021.0223
Y397F TM7 Impair β-arrestin signaling, with no effect on G-protein signaling https://doi.org/10.1089/can.2021.0223
I156A TM2 Enhances β-arrestin1 recruitment https://doi.org/10.1089/can.2021.0223
I156T TM2 Reduces β-arrestin recruitment https://doi.org/10.1089/can.2021.0223
D163E TM2 Loss of G-protein signaling https://doi.org/10.1016/j.sbi.2019.04.007
D163N TM2 Loss of G-protein signaling https://doi.org/10.1016/j.sbi.2019.04.007
K192E TM3 Attenuates receptor activation https://doi.org/10.1016/j.sbi.2019.04.007
C257A ECL2 No ligand binding https://doi.org/10.1016/j.sbi.2019.04.007
C264A ECL2 No ligand binding https://doi.org/10.1016/j.sbi.2019.04.007
Y275F TM5 cAMP inhibition slightly reduced https://doi.org/10.1016/j.sbi.2019.04.007
M363A TM6 Reduced G-protein and ligand binding https://doi.org/10.1016/j.sbi.2019.04.007
W356A TM6 Enhanced agonist activation https://doi.org/10.1038%2Fs41598-021-01767-5
D164N TM2 Blocks coupling of the receptor to the potentiation of inwardly rectifying potassium channel (KIR) currents and prevents internalization of the receptor after exposure to agonist https://doi.org/10.1124/mol.56.3.611
Y275F TM5 Subtle differences in WT binding and signal transduction https://doi.org/10.1016/s0006-2952(02)01031-6
M363A TM6 Diminished HU210 binding https://doi.org/10.1074/jbc.m111.261651
Q115A TM1 Weak ligand binding affinity https://doi.org/10.1016/s0006-2952(03)00155-2
L193A TM3 Diminished HU210 binding https://doi.org/10.1074/jbc.m111.261651
Y275I TM5 Receptors that could not produce signal transduction or bind to multiple cannabinoid compounds https://doi.org/10.1016/s0006-2952(02)01031-6
C386A TM7 Impairs antagonist binding https://doi.org/10.1021/bi0472651
F174A TM2 Diminished HU210 binding https://doi.org/10.1074/jbc.m111.261651
F177A TM2 Diminished HU210 binding https://doi.org/10.1074/jbc.m111.261651
V282F TM5 Increases the affinity of WIN55212-2 for CB(1) by 12-fold https://pubmed.ncbi.nlm.nih.gov/10496968/
C264S ECL2 Eliminated CP 55,940 binding https://doi.org/10.1074/jbc.271.12.6941
S114A TM1 Weak ligand binding affinity https://doi.org/10.1016/s0006-2952(03)00155-2
C386M TM7 Impairs antagonist binding https://doi.org/10.1021/bi0472651
C257A ECL2 Functionality of the receptor is lost https://doi.org/10.1021/bi0472651
K192A TM3 SR 141716A appeared to become a neutral antagonist at the K192A mutant receptor https://doi.org/10.1124/mol.54.6.1064
G195S TM3 Enhancement in WIN 55,212-2 binding https://pubmed.ncbi.nlm.nih.gov/10525107/
C257S ECL2 Eliminated CP 55,940 binding https://doi.org/10.1074/jbc.271.12.6941


Mutations in Cannabinoid receptor 2
Residue Position Transmembrane Region Effect References
K109A TM3 No effect on agonist binding. Affects cannabinoid agonist binding; when associated with G-112. https://pubmed.ncbi.nlm.nih.gov/10051546/
S112G TM3 Affects cannabinoid agonist binding; when associated with A-109. https://pubmed.ncbi.nlm.nih.gov/10051546/
L201P TM5 Complete loss of the ability of cannabinoid agonists to inhibit forskolin-stimulated cAMP accumulation. https://doi.org/10.1016/s0014-5793(02)03537-8
Y209A TM5 Complete loss of the ability of cannabinoid agonists to inhibit forskolin-stimulated cAMP accumulation. https://doi.org/10.1016/s0014-5793(02)03537-8
D130A TM3 Abolished constitutive activity of the wild-type CB2 receptor,abolished ligand binding https://doi.org/10.1016/s0006-2952(03)00005-4
R131A TM3 Abolished constitutive activity of the wild-type CB2 receptor https://doi.org/10.1016/s0006-2952(03)00005-4
A244E TM6 Abolished constitutive activity of the wild-type CB2 receptor,abolished ligand binding https://doi.org/10.1016/s0006-2952(03)00005-4
W158Y TM4 Completely lost ligand binding capacity https://doi.org/10.1046/j.1471-4159.2000.0752485.x
W172F ECL2 Retained cannabinoid binding and downstream signaling (inhibition of adenylyl cyclase) https://doi.org/10.1046/j.1471-4159.2000.0752485.x
C179S ECL2 Eliminate ligand binding https://doi.org/10.1074/jbc.271.12.6941
W172L ECL2 Retained cannabinoid binding and downstream signaling (inhibition of adenylyl cyclase) https://doi.org/10.1046/j.1471-4159.2000.0752485.x
W158A TM4 Completely lost ligand binding capacity https://doi.org/10.1046/j.1471-4159.2000.0752485.x
W158F TM4 Retained wild-type binding and signaling activities https://doi.org/10.1046/j.1471-4159.2000.0752485.x
R131A TM3 Partially reduced the cannabinoid-induced inhibition of adenylyl cyclase https://doi.org/10.1016/s0014-5793(00)01094-2
F197V TM5 Decreases the affinity of ligand for CB(2) by 14-fold https://pubmed.ncbi.nlm.nih.gov/10496968/
Y299A TM7 Complete loss of ligand binding and a severe impairment of cannabinoid-induced inhibition of forskolin-stimulated cAMP accumulation https://doi.org/10.1016/s0014-5793(01)02642-4
W172Y ECL2 Retained cannabinoid binding and downstream signaling (inhibition of adenylyl cyclase) https://doi.org/10.1046/j.1471-4159.2000.0752485.x
V113E TM3 Complete loss of CB2 ligand binding as well as downstream signaling activities https://doi.org/10.1016/j.bbrc.2014.08.048
L192S TM5 Complete loss of CB2 ligand binding as well as downstream signaling activities https://doi.org/10.1016/j.bbrc.2014.08.048
W172A ECL2 Eliminated agonist binding https://doi.org/10.1046/j.1471-4159.2000.0752485.x
D130A TM3 Reduced binding of cannabinoid agonists https://doi.org/10.1016/s0014-5793(00)01094-2
C175S ECL2 Wild-type receptor properties with CP 55,940, loss of SR 144528 binding and eight-fold reduced binding and activity of WIN 55212-2 https://doi.org/10.1016/s0014-2999(00)00439-8
Y132A TM3 Reduced the cannabinoid-induced inhibition of adenylyl cyclase https://doi.org/10.1016/s0014-5793(00)01094-2
C313A C-ter Reduced coupling to AC https://doi.org/10.1016/S0014-5793(01)02642-4