title: Differential regulation of corticotropin releasing factor 1alpha receptor endocytosis and trafficking by beta-arrestins and Rab GTPases.
abstract: The corticotropin releasing factor (CRF) type 1alpha receptor, a member of the G protein-coupled receptor (GPCR) subfamily B, is involved in the aetiology of anxiety and depressive disorders. In the present study, we examined the internalization and trafficking of the CRF1alpha receptor in both human embryonic kidney (HEK)293 cells and primary cortical neurons. We found that CRF1alpha receptor activation leads to the selective recruitment of beta-arrestin2 in both HEK293 cells and neurons. We observed distinct distribution patterns of CRF1alpha receptor and beta-arrestin2 in HEK293 cells and cortical neurons. In HEK293 cells, beta-arrestin2-green fluorescent protein (GFP) co-localized with CRF1alpha receptor in vesicles at the plasma membrane but was dissociated from the receptor in endosomes. In contrast, in primary cortical neurons, beta-arrestin2 and CRF1alpha receptor were internalized in distinct endocytic vesicles. By bioluminescence resonance energy transfer, we demonstrated that beta-arrestin2 association with CRF1alpha receptor was increased in cells transfected with G protein-coupled receptor kinase (GRK)3 and GRK6 and decreased in cells transfected with GRK2 and GRK5. In both HEK293 cells and cortical neurons, internalized CRF1alpha receptor transited from Rab5-positive early endosomes to Rab4-positive recycling endosomes and was not targeted to lysosomes. However, CRF1alpha receptor resensitization was blocked by the overexpression of wild-type, but not dominant-negative, Rab5 and Rab4 GTPases. Taken together, our results suggest that beta-arrestin trafficking differs between HEK293 cells and neurons, and that CRF1alpha receptor resensitization is regulated in an atypical manner by Rab GTPases.
authors.affiliation: Cell Biology Research Group, Robarts Research Institute, University of Western Ontario, London, Ontario, Canada.
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Holmes
authors.forename: Andy V
authors.initials: AV
authors.lastname: Babwah
authors.forename: Lianne B
authors.initials: LB
authors.lastname: Dale
authors.forename: Michael O
authors.initials: MO
authors.lastname: Poulter
authors.forename: Stephen S G
authors.initials: SS
authors.lastname: Ferguson
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D019390:Arrestins / Q000502:physiology*; D002460:Cell Line; D002540:Cerebral Cortex / Q000502:physiology; D004705:Endocytosis; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007668:Kidney; D007700:Kinetics; D049449:Luminescence; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000502:physiology; D018019:Receptors, Corticotropin-Releasing Hormone / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D000071557:beta-Arrestins; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D000099052:CRF Receptor, Type 1
chemical_list: D019390:Arrestins; D018019:Receptors, Corticotropin-Releasing Hormone; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000071557:beta-Arrestins; D049452:Green Fluorescent Proteins; D000099052:CRF Receptor, Type 1; D020691:rab GTP-Binding Proteins; D000262:Adenylyl Cyclases