Internalization and Recycling Pathways of the Thyrotropin Receptor

215

234

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.

Section: Cb1-egfp Is Functional and Displays A Predominantly Intracelsupporting

confidence: 80%

“…If AM281 acts by sequestering CB1Rs that have been delivered to the plasma membrane by constitutive recycling, blocking the recycling process would antagonize the AM281 effect. Monensin, a potent inhibitor of recycling (11,25), strongly inhibits the AM281-induced externalization (Fig. 5C).…”

Section: Cb1r Is Constitutively Endocytosed and Am281-induced Externamentioning

confidence: 90%

Constitutive Endocytic Cycle of the CB1 Cannabinoid Receptor

Leterrier

Bonnard

Carrel

et al. 2004

215

234

“…This is further supported by the observation that a specific arrestin interacting motif in the GPCR carboxylterminal tail dictates the rate of receptor dephosphorylation, recycling, and resensitization (36). Moreover, the carboxyl-terminal tails of some GPCRs have a great influence on the sorting of these internalized GPCRs to recycling pathways (37,38), and even two distinct structural motifs have been identified to direct the sorting of internalized hormone receptor from degradation to recycling (39). Taken together, we propose that the mutual interaction between specific motif(s) in the C terminus of the wild type MOR and ␤-arrestin, which is promoted by phosphorylation, directs the internalized receptors to recycling pathway.…”

Section: Discussionmentioning

confidence: 84%

μ-Opioid Receptor Desensitization

Qiu

Law

Loh

2003

It is generally accepted that the internalization and desensitization of -opioid receptor (MOR) involves receptor phosphorylation and ␤-arrestin recruitment. However, a mutant MOR, which is truncated after the amino acid residue Ser 363 (MOR363D), was found to undergo phosphorylation-independent internalization and desensitization. As expected, MOR363D, missing the putative agonist-induced phosphorylation sites, did not exhibit detectable agonist-induced phosphorylation. MOR363D underwent slower internalization as reflected in the attenuation of membrane translocation of ␤-arrestin 2 when compared with wild type MOR, but the level of receptor being internalized was similar to that of wild type MOR after 4 h of etorphine treatment. Furthermore, MOR363D was observed to desensitize faster than that of wild type MOR upon agonist activation. Surface biotinylation assay demonstrated that the wild type receptors recycled back to membrane after agonist-induced internalization, which contributed to the receptor resensitization and thus partially reversed the receptor desensitization. On the contrary, MOR363D did not recycle after internalization. Hence, MOR desensitization is controlled by the receptor internalization and the recycling of internalized receptor to cell surface in an active state. Taken together, our data indicated that receptor phosphorylation is not absolutely required in the internalization, but receptor phosphorylation and subsequent ␤-arrestin recruitment play important roles in the resensitization of internalized receptors.The regulation of G protein-coupled receptors (GPCRs) 1 signaling highlights the complex relationship between multiple mechanisms acting at different levels of signal propagation. Following the agonist activation of the receptor, G proteincoupled receptor kinases (GRKs) are recruited to the membrane and phosphorylate the agonist-activated GPCRs. GRKmediated receptor phosphorylation enhances the interaction between GPCRs and cytoplasmic proteins, arrestins, which uncouple the GPCRs from G proteins and also target the GPCRs to clathrin-coated pits for internalization (endocytosis). The internalized receptors can subsequently be dephosphorylated and recycled back to the plasma membrane, which contributes to resensitization. Alternatively, the internalized receptors can be targeted to lysosome for degradation (1-4). Receptor desensitization, the progressive loss of receptor function under continued exposure to an agonist, occurs in many GPCRs. Desensitization can be a consequence of multiple processes of receptor uncoupling, internalization, degradation, and recycling. Therefore, phosphorylation of agonist-activated receptors and subsequent arrestin recruitment are important processes in the modulation of GPCRs responsiveness.However, multiple studies challenge the critical role phosphorylation played in desensitization and internalization of many GPCRs. Desensitization and internalization of human parathyroid hormone receptor (5), AT 1A angiotensin receptor (6), and metabotropic gluta...

“…Thus, it is now known that the complex formed by the rat, mouse, or porcine LHR and one of its agonists (hCG) is internalized via clathrincoated pits (53) by a pathway that requires the involvement of a non-visual arrestin and dynamin (29,51). The rodent or porcine LHR⅐hCG complex is resistant to dissociation by the mild acidic pH that prevails in the endosomes (50), and a substantial proportion of the internalized complex is routed to the lysosomes where it dissociates prior to degradation (50,(52)(53)(54). By promoting the accumulation of the hCG⅐LHR complex in a compartment where it can be degraded, this pathway is ultimately responsible not only for the degradation of hCG (28) but also for a substantial loss of cell surface LHR that ensues following exposure of target or heterologous cells expressing the rodent or porcine LHR to agonists (13,36,55,56).…”

Section: Resultsmentioning

confidence: 99%

GIPC Binds to the Human Lutropin Receptor (hLHR) through an Unusual PDZ Domain Binding Motif, and It Regulates the Sorting of the Internalized Human Choriogonadotropin and the Density of Cell Surface hLHR

Hirakawa

Galet

Kishi

et al. 2003

By using a yeast two-hybrid screen we identified GIPC (GAIP-interacting protein C terminus), a protein with a type I PDZ domain as a novel human lutropin receptor (hLHR) binding partner. Pull-down and immunoprecipitation assays confirmed this interaction and showed that it is dependent on the PDZ domain of GIPC and the C-terminal tetrapeptide of the hLHR. To characterize the functional consequences of the GIPC-hLHR interaction, we used a small interfering RNA against GIPC to generate a clonal cell line that is deficient in GIPC. Studies with this cell line reveal that GIPC is partially responsible for the recycling of the hormone that is internalized by the hLHR and also for maintaining a relatively constant level of hLHR at the cell surface during hormone internalization.