title: Visualization of agonist-induced association and trafficking of green fluorescent protein-tagged forms of both beta-arrestin-1 and the thyrotropin-releasing hormone receptor-1.
abstract: A fusion protein (beta-arrestin-1-green fluorescent protein (GFP)) was constructed between beta-arrestin-1 and a modified form of the green fluorescent protein from Aequorea victoria. Expression in HEK293 cells allowed immunological detection of an 82-kDa cytosolic polypeptide with antisera to both beta-arrestin-1 and GFP. Transient expression of this construct in HEK293 cells stably transfected to express the rat thyrotropin-releasing hormone receptor-1 (TRHR-1) followed by confocal microscopy allowed its visualization evenly distributed throughout the cytoplasm. Addition of thyrotropin-releasing hormone (TRH) caused a profound and rapid redistribution of beta-arrestin-1-GFP to the plasma membrane followed by internalization of beta-arrestin-1-GFP into distinct, punctate, intracellular vesicles. TRH did not alter the cellular distribution of GFP transiently transfected into these cells nor the distribution of beta-arrestin-1-GFP following expression in HEK293 cells lacking the receptor. To detect potential co-localization of the receptor and beta-arrestin-1 in response to agonist treatment, beta-arrestin-1-GFP was expressed stably in HEK293 cells. A vesicular stomatitis virus (VSV)-tagged TRHR-1 was then introduced transiently. Initially, the two proteins were fully resolved. Short term exposure to TRH resulted in their plasma membrane co-localization, and sustained exposure to TRH resulted in their co-localization in punctate, intracellular vesicles. In contrast, beta-arrestin-1-GFP did not relocate or adopt a punctate appearance in cells that did not express VSV-TRHR-1. Reciprocal experiments were performed, with equivalent results, following transient expression of beta-arrestin-1 into cells stably expressing VSVTRHR-1-GFP. These results demonstrate the capacity of beta-arrestin-1-GFP to interact with the rat TRHR-1 and directly visualizes their recruitment from cytoplasm and plasma membrane respectively into overlapping, intracellular vesicles in an agonist-dependent manner.
authors.affiliation: Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, Davidson Building, University of Glasgow, Glasgow G12 8QQ, United Kingdom.
authors.forename: D A
authors.initials: DA
authors.lastname: Groarke
authors.forename: S
authors.initials: S
authors.lastname: Wilson
authors.forename: C
authors.initials: C
authors.lastname: Krasel
authors.forename: G
authors.initials: G
authors.lastname: Milligan
mesh_terms: D000818:Animals; D019390:Arrestins / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers; D004705:Endocytosis; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D011485:Protein Binding; D051381:Rats; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000819:agonists / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins
chemical_list: C000605253:ARRB1 protein, human; C000605256:Arrb1 protein, rat; D019390:Arrestins; D017931:DNA Primers; D008164:Luminescent Proteins; D018025:Receptors, Thyrotropin-Releasing Hormone; D011993:Recombinant Fusion Proteins; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins; D049452:Green Fluorescent Proteins