Role of phospholipase D in parathyroid hormone type 1 receptor signaling and trafficking.
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.
Garrido
José Luis
JL
Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pennsylvania 15261, USA.
Wheeler
David
D
Vega
Luis Leiva
LL
Friedman
Peter A
PA
Romero
Guillermo
G
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*
D010281:Parathyroid Hormone; D044168:Receptor, Parathyroid Hormone, Type 1; C105606:phospholipase D2; D010739:Phospholipase D; C106801:phospholipase D1