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Regulation of CB1 cannabinoid receptor internalization by a promiscuous phosphorylation-dependent mechanism.

ABSTRACT: Agonists stimulate cannabinoid 1 receptor (CB(1)R) internalization. Previous work suggests that the extreme carboxy-terminus of the receptor regulates this internalization - likely through the phosphorylation of serines and threonines clustered within this region. While truncation of the carboxy-terminus (V460Z CB(1)) and consequent removal of these putative phosphorylation sites prevents endocytosis in AtT20 cells, the residues necessary for CB(1)R internalization remain elusive. To determine the structural requirements for internalization, we evaluated endocytosis of carboxy-terminal mutant CB(1)Rs stably expressed in HEK293 cells. In contrast to AtT20 cells, V460Z CB(1)R expressed in HEK293 cells internalized to the same extent and with similar kinetics as the wild-type receptor. However, mutation of serine and/or threonine residues within the extreme carboxy-terminal attenuated internalization when these receptors were expressed in HEK293 cells. These results establish that the extreme carboxy-terminal phosphorylation sites are not required for internalization of truncated receptors, but are required for internalization of full-length receptors in HEK293 cells. Analysis of beta-arrestin-2 recruitment to mutant CB(1)R suggests that putative carboxy-terminal phosphorylation sites mediate beta-arrestin-2 translocation. This study indicates that the local cellular environment affects the structural determinants of CB(1)R internalization. Additionally, phosphorylation likely regulates the internalization of (full-length) CB(1)Rs.

SUBMITTER: Daigle TL

PROVIDER: S-EPMC3707135 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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Regulation of CB1 cannabinoid receptor internalization by a promiscuous phosphorylation-dependent mechanism.

Daigle Tanya L TL Kwok Mary Lee ML Mackie Ken K

Journal of neurochemistry 20080701 1

Agonists stimulate cannabinoid 1 receptor (CB(1)R) internalization. Previous work suggests that the extreme carboxy-terminus of the receptor regulates this internalization - likely through the phosphorylation of serines and threonines clustered within this region. While truncation of the carboxy-terminus (V460Z CB(1)) and consequent removal of these putative phosphorylation sites prevents endocytosis in AtT20 cells, the residues necessary for CB(1)R internalization remain elusive. To determine t ...[more]

PMID: 18331587

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Project description:The cannabinoid CB(1) (CB(1)) and dopamine D(2) (D(2)) receptors are coexpressed in the basal ganglia, an area of the brain involved in such processes as cognition, motor function, and emotional control. Several lines of evidence suggest that CB(1) and D(2) receptors may oligomerize, providing a unique pharmacology in vitro and in vivo. However, limited information exists on the regulation of CB(1) and D(2) receptor dimers. We used a novel technique, multicolor bimolecular fluorescence complementation (MBiFC) to examine the subcellular localization of CB(1)-D(2L) heterodimers as well as D(2L)-D(2L) homodimers in a neuronal cell model, Cath. a differentiated cells. MBiFC was then used to explore the effects of persistent ligand treatment on receptor dimerization at the plasma membrane and intracellularly. Persistent (20-h) agonist treatment resulted in increased formation of CB(1)-D(2L) heterodimers relative to the D(2L)-D(2L) homodimers. The effects of the D(2) agonist quinpirole were restricted to the intracellular compartment and may reflect increased D(2L) receptor expression. In contrast, treatment with the CB(1) receptor agonist (2)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol (CP55, 940) produced increases in both membrane and intracellular CB(1)-D(2L) heterodimers independently of alterations in CB(1) receptor expression. The effects of CB(1) receptor activation were attenuated by the CB(1) antagonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide (AM281) and were both time- and dose-dependent. The effects of CB(1) activation were examined further by combining MBiFC with a constitutively active CB(1) receptor mutant, CB(1)T210I. These studies demonstrated that the expression of CB(1)T210I increased intracellular CB(1)-D(2L) heterodimer formation. In summary, agonist-induced modulation of CB(1)-D(2L) oligomerization may have physiological implications in diseases such as Parkinson's disease and drug abuse.

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Dataset Information

Regulation of CB1 cannabinoid receptor internalization by a promiscuous phosphorylation-dependent mechanism.

Publications

Regulation of CB1 cannabinoid receptor internalization by a promiscuous phosphorylation-dependent mechanism.

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