Structural domains of the CB1 cannabinoid receptor that contribute to constitutive activity and G-protein sequestration.
The CB1 cannabinoid receptor is a constitutively active receptor that can sequester G(i/o)-proteins and prevent other G(i/o)-coupled receptors from signaling (Bouaboula et al., 1997; Pan et al., 1998; Vasquez and Lewis, 1999). G-protein sequestration occurs because the population of CB1 cannabinoid receptors exists in both an inactive G-protein-precoupled RG(GDP) state and a constitutively active R*G(GTP) state. We tested the hypothesis that the distal C-terminal tail acts to prevent G-protein activation. We found that truncation of the distal C-terminal tail of the CB1 receptor (CB1-417) enhanced both the constitutive activity and the ability of the receptor to sequester G-proteins. In addition, we tested the hypothesis that the conserved aspartate (D2.50) in the second transmembrane domain of the CB1 cannabinoid receptor is crucial for constitutive activity and G-protein sequestration. We found that the mutation of aspartate to asparagine (CB1-D164N) abolished G-protein sequestration and constitutive receptor activity without disrupting agonist-stimulated activity. We conclude that the CB1-D164N mutation and the C-terminal truncation shift the population of receptors in opposite directions. The CB1-D164N mutation shifts the receptor into an inactive R state upcoupled from G-proteins, whereas the C-terminal truncation (CB1-417) shifts the receptor into the active R*G(GTP) state. Thus the distal C-terminal tail acts to constrain the receptor from activating G-proteins, whereas the aspartate (D2.50) in the second transmembrane domain stabilizes the receptor in both the inactive RG(GDP) state and the active R*G(GTP) state.
Nie
J
J
Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia 30912, USA.
Lewis
D L
DL
D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D019204:GTP-Binding Proteins / Q000378:metabolism*; D018014:Gene Transfer Techniques; D006801:Humans; D008297:Male; D008845:Microinjections; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D017783:Superior Cervical Ganglion / Q000166:cytology / Q000187:drug effects / Q000378:metabolism
D043882:Receptors, Cannabinoid; D011955:Receptors, Drug; D019204:GTP-Binding Proteins; D002118:Calcium