title: Fluorescent ligand-directed co-localization of the parathyroid hormone 1 receptor with the brush-border scaffold complex of the proximal tubule reveals hormone-dependent changes in ezrin immunoreactivity consistent with inactivation.
abstract: Through binding to parathyroid hormone (PTH), PTH1R interacts with kidney-specific scaffold proteins, including the sodium hydrogen exchanger regulatory factors 1 and 2 (NHERFs), and ezrin. To facilitate in vivo localization, tetramethylrhodamine-labeled PTH (PTH-TMR) was used as a fluorescent probe. In mice, PTH-TMR localizes to luminal surfaces of tubular S1 segments that overlap PTH1R immunostaining, but does not directly overlap with megalin-specific antibodies. PTH-TMR staining directly overlaps with Npt2a in nascent, endocytic vesicles, marking the location of transporter regulation. PKA substrate antibodies display marked staining increases in segments labeled with PTH-TMR, demonstrating a functional effect. In the presence of secondary hyperparathyroidism, PTH-TMR staining is markedly reduced and shifts to co-localizing with megalin. At 15min post-injection, PTH-TMR-labeled vesicles do not co-localize with either NHERF or ezrin, suggesting PTH1R dissociation from the scaffold complex. At the 5min time point, PTH-TMR stains the base of microvilli where it localizes with both NHERF2 and ezrin, and only partially with NHERF1. Strikingly, the bulk of ezrin protein becomes undetectable with the polyclonal, CS3145 antibody, revealing a PTH-induced conformational change in the scaffold. A second ezrin antibody (3C12) is capable of detecting the altered ezrin protein. The CS3145 antibody only binds to the active form of ezrin and fails to recognize the inactive form, while the 3C12 reagent can detect either active or inactive ezrin. Here we show that the PTH1R is part of the ezrin scaffold complex and that acute actions of PTH suggest a rapid inactivation of ezrin in a spatially defined manner.
authors.affiliation: Massachusetts General Hospital and Department of Medicine, Harvard Medical School, Boston, MA 02114, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Guo
authors.forename: Lige
authors.initials: L
authors.lastname: Song
authors.forename: Minlin
authors.initials: M
authors.lastname: Liu
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Mahon
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006575:Heterocyclic Compounds, 3-Ring; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008871:Microvilli / Q000187:drug effects / Q000378:metabolism*; D010281:Parathyroid Hormone / Q000494:pharmacology*; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000378:metabolism*; D012235:Rhodamines; D000099149:Ezrin
chemical_list: D003598:Cytoskeletal Proteins; D006575:Heterocyclic Compounds, 3-Ring; D010281:Parathyroid Hormone; D044168:Receptor, Parathyroid Hormone, Type 1; D012235:Rhodamines; D000099149:Ezrin; C412715:tetramethylrosamine