Abstract
The major effects of cannabinoids and endocannabinoids are mediated via two G protein-coupled receptors, CB1 and CB2, elucidation of the mechanism and structural determinants of the CB2 receptor coupling with G proteins will have a significant impact on drug discovery. In the present study, we systematically investigated the role of the intracellular loops in the interaction of the CB2 receptor with G proteins using chimeric receptors alongside the characterization of cAMP accumulation and ERK1/2 phosphorylation. We provided evidence that ICL2 was significantly involved in G protein coupling in coordination with the C-terminal end. Moreover, a single alanine substitution of the Pro-139 in the CB2 receptor that corresponds to Leu-222 in the CB1 receptor resulted in a moderate impairment in the inhibition of cAMP accumulation, whereas mutants P139F, P139M and P139L were able to couple to the Gs protein in a CRE-driven luciferase assay. With the ERK activation experiments, we further found that P139L has the ability to activate ERK through both Gi- and Gs-mediated pathways. Our findings defined an essential role of the second intracellular loop of the CB2 receptor in coordination with the C-terminal tail in G protein coupling and receptor activation.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenylyl Cyclase Inhibitors
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Adenylyl Cyclases / metabolism
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Amino Acid Sequence
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Cell Membrane / drug effects
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Cell Membrane / metabolism
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Cyclic AMP-Dependent Protein Kinases / antagonists & inhibitors
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Cyclic AMP-Dependent Protein Kinases / metabolism
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Enzyme Activation / drug effects
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Extracellular Signal-Regulated MAP Kinases / metabolism
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GTP-Binding Proteins / metabolism*
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HEK293 Cells
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Humans
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Molecular Sequence Data
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Mutant Proteins / chemistry
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Mutant Proteins / metabolism
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Proline / metabolism
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Protein Binding / drug effects
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Protein Kinase Inhibitors / pharmacology
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Protein Structure, Secondary
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Protein Structure, Tertiary
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Receptor, Cannabinoid, CB1 / chemistry
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Receptor, Cannabinoid, CB1 / metabolism
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Receptor, Cannabinoid, CB2 / agonists
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Receptor, Cannabinoid, CB2 / chemistry*
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Receptor, Cannabinoid, CB2 / metabolism*
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Recombinant Proteins / metabolism
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Signal Transduction* / drug effects
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Structure-Activity Relationship
Substances
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Adenylyl Cyclase Inhibitors
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Mutant Proteins
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Protein Kinase Inhibitors
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Receptor, Cannabinoid, CB1
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Receptor, Cannabinoid, CB2
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Recombinant Proteins
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Proline
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Cyclic AMP-Dependent Protein Kinases
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Extracellular Signal-Regulated MAP Kinases
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GTP-Binding Proteins
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Adenylyl Cyclases