Using green fluorescent proteins to study G-protein-coupled receptor localization and trafficking

Kallal, Lorena A.; Benovic, Jeffrey L.

doi:10.1016/s0165-6147(00)01477-2

Cited by 109 publications

(86 citation statements)

References 52 publications

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“…Such fusion proteins are now widely used to study protein trafficking (38), and their usefulness is amply demonstrated for GPCRs (39,40). In our study, we first verified that CB1-EGFP was functional by measuring constitutive inhibition of cAMP accumulation after forskolin stimulation, showing that tagged CB1-EGFP and untagged CB1-WT behaved similarly.…”

Section: Cb1r Is Constitutively Endocytosed and Am281-induced Externasupporting

confidence: 60%

Constitutive Endocytic Cycle of the CB1 Cannabinoid Receptor

Leterrier

Bonnard

Carrel

et al. 2004

Journal of Biological Chemistry

214

234

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The CB1 cannabinoid receptor (CB1R) displays a significant level of ligand-independent (i.e. constitutive) activity, either when heterologously expressed in nonneuronal cells or in neurons where CB1Rs are endogenous. The present study investigates the consequences of constitutive activity on the intracellular trafficking of CB1R. When transfected in HEK-293 cells, CB1R is present at the plasma membrane, but a substantial proportion (ϳ85%) of receptors is localized in intracellular vesicles. Detailed analysis of CB1-EGFP expressed in HEK-293 cells shows that the intracellular CB1R population is mostly of endocytic origin and that treatment with inverse agonist AM281 traps CB1R at the plasma membrane through a monensin-sensitive recycling pathway. Co-transfection with dominant positive or dominant negative mutants of the small GTPases Rab5 and Rab4, but not Rab11, profoundly modifies the steady-state and ligand-induced intracellular distribution of CB1R, indicating that constitutive endocytosis is Rab5-dependent, whereas constitutive recycling is mediated by Rab4. In conclusion, our results indicate that, due to its natural constitutive activity, CB1R permanently and constitutively cycles between plasma membrane and endosomes, leading to a predominantly intracellular localization at steady state.

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Section: Cb1r Is Constitutively Endocytosed and Am281-induced Externasupporting

confidence: 60%

Constitutive Endocytic Cycle of the CB1 Cannabinoid Receptor

Leterrier

Bonnard

Carrel

et al. 2004

Journal of Biological Chemistry

214

234

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“…G-protein-coupled receptors are the single largest family of cell surface receptors involved in signal transduction and represent major targets for the development of novel drug candidates in all clinical areas (Bikker et al, 1998). It is estimated that 30% of clinically prescribed drugs function as either agonists or antagonists of G-protein-coupled receptors which emphasizes their immense therapeutic potential (Kallal and Benovic, 2000;Stadel et al, 1997).…”

Section: Discussionmentioning

confidence: 99%

Interaction of Serotonin_1AReceptors from Bovine Hippocampus with Tertiary Amine Local Anesthetics

Kalipatnapu

Chattopadhyay

2004

Cell Mol Neurobiol

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SUMMARY1. We have examined the interaction of tertiary amine local anesthetics with the bovine hippocampal serotonin 1A (5-HT 1A ) receptor, an important member of the G-proteincoupled receptor superfamily.2. The local anesthetics inhibit specific agonist and antagonist binding to the 5-HT 1A receptor at a clinically relevant concentration range of the anesthetics. This is accompanied by a concomitant reduction in the binding affinity of the 5-HT 1A receptor to the agonist. Interestingly, the extent of G-protein coupling of the receptor is reduced in the presence of the local anesthetics.3. Fluorescence polarization measurements using depth-dependent fluorescent probes show that procaine and lidocaine do not show any significant change in membrane fluidity. On the other hand, tetracaine and dibucaine were found to alter fluidity of the membrane as indicated by a fluorescent probe which monitors the headgroup region of the membrane.4. The local anesthetics showed inhibition of agonist binding to the 5-HT 1A receptor in membranes depleted of cholesterol more or less to the same extent as that of control membranes in all cases. This suggests that the inhibition in ligand binding to the 5-HT 1A receptor brought about by local anesthetics is independent of the membrane cholesterol content.5. Our results on the effects of the local anesthetics on the ligand binding and G-protein coupling of the 5-HT 1A receptor support the possibility that G-protein-coupled receptors could be involved in the action of local anesthetics.

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“…For stable transfections, HEK 293 cells were seeded at 1.5 ϫ 10 6 cells/100-mm dish and transfected the following day with 10 g of DNA by liposome-mediated transfer (DOTAP), as described previously (25,26). Stable transfectants were selected in the presence of G418 (0.5-1.5 mg/ml) and/or hygromycin (50 -75 g/ml).…”

Section: Methodsmentioning

confidence: 99%

Dimerization of the Human Receptors for Prostacyclin and Thromboxane Facilitates Thromboxane Receptor-mediated cAMP Generation

Wilson

Roche

Kostetskaia

et al. 2004

Journal of Biological Chemistry

157

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Using green fluorescent proteins to study G-protein-coupled receptor localization and trafficking

Cited by 109 publications

References 52 publications

Constitutive Endocytic Cycle of the CB1 Cannabinoid Receptor

Constitutive Endocytic Cycle of the CB1 Cannabinoid Receptor

Interaction of Serotonin_1AReceptors from Bovine Hippocampus with Tertiary Amine Local Anesthetics

Dimerization of the Human Receptors for Prostacyclin and Thromboxane Facilitates Thromboxane Receptor-mediated cAMP Generation

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Using green fluorescent proteins to study G-protein-coupled receptor localization and trafficking

Cited by 109 publications

References 52 publications

Constitutive Endocytic Cycle of the CB1 Cannabinoid Receptor

Constitutive Endocytic Cycle of the CB1 Cannabinoid Receptor

Interaction of Serotonin1AReceptors from Bovine Hippocampus with Tertiary Amine Local Anesthetics

Dimerization of the Human Receptors for Prostacyclin and Thromboxane Facilitates Thromboxane Receptor-mediated cAMP Generation

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Interaction of Serotonin_1AReceptors from Bovine Hippocampus with Tertiary Amine Local Anesthetics