title: Role of phospholipase D in parathyroid hormone type 1 receptor signaling and trafficking.
abstract: The role of phospholipase D (PLD) in the regulation of the traffic of the PTH type 1 receptor (PTH1R) was studied in Chinese hamster ovary cells stably transfected with a human PTH1R (CHO-R3) and in rat osteosarcoma 17/2.8 (ROS) cells. PTH(1-34) increased total PLD activity by 3-fold in CHO-R3 cells and by 2-fold in ROS cells. Overexpression of wild-type (WT) PLD1 and WT-PLD2 increased basal PLD activity in CHO-R3 but not in ROS cells. Ligand-stimulated PLD activity greatly increased in CHO-R3 cells transfected with WT-PLD1 and WT-PLD2. However, only WT-PLD2 expression increased PTH-dependent PLD activity in ROS cells. Expression of the catalytically inactive mutants R898K-PLD1 (DN-PLD1) and R758K-PLD2 (DN-PLD2) inhibited ligand-dependent PLD activity in both cell lines. PTH(1-34) induced internalization of the PTH1R with a concomitant increase in the colocalization of the receptor with PLD1 in intracellular vesicles and in a perinuclear, ADP ribosylation factor-1-positive compartment. The distribution of PLD1 and PLD2 remained unaltered after PTH treatment. Expression of DN-PLD1 had a small effect on endocytosis of the PTH1R; however, DN-PLD1 prevented accumulation of the PTH1R in the perinuclear compartment. Expression of DN-PLD2 significantly retarded ligand-induced PTH1R internalization in both cell lines. The differential effects of PLD1 and PLD2 on receptor traffic were confirmed using isoform-specific short hairpin RNA constructs. We conclude that PLD1 and PLD2 play distinct roles in regulating PTH1R traffic; PLD2 primarily regulates endocytosis, whereas PLD1 regulates receptor internalization and intracellular receptor traffic.
authors.affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pennsylvania 15261, USA.
authors.forename: José Luis
authors.initials: JL
authors.lastname: Garrido
authors.forename: David
authors.initials: D
authors.lastname: Wheeler
authors.forename: Luis Leiva
authors.initials: LL
authors.lastname: Vega
authors.forename: Peter A
authors.initials: PA
authors.lastname: Friedman
authors.forename: Guillermo
authors.initials: G
authors.lastname: Romero
mesh_terms: D000818:Animals; D016466:CHO Cells; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D010281:Parathyroid Hormone / Q000494:pharmacology; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
chemical_list: D010281:Parathyroid Hormone; D044168:Receptor, Parathyroid Hormone, Type 1; C105606:phospholipase D2; D010739:Phospholipase D; C106801:phospholipase D1