Parathyroid hormone receptor recycling: role of receptor dephosphorylation and beta-arrestin.
The recovery of PTH receptor (PTHR) function after acute homologous receptor desensitization and down-regulation in bone and kidney cells has been attributed to receptor recycling. To determine the role of receptor dephosphorylation in PTHR recycling, we performed morphological and functional assays on human embryonic kidney 293 cells stably expressing wild-type (wt) or mutant PTHRs. Confocal microscopy and ligand binding assays revealed that the wt PTHR is rapidly recycled back to the plasma membrane after removal of the agonist. Receptors that were engineered to either lack the sites of phosphorylation or to resemble constitutively phosphorylated receptors were able to recycle back to the plasma membrane with the same kinetics as the wt PTHR. The PTHR was found to be dephosphorylated by an enzyme apparently distinct from protein phosphatases 1 or 2A. The PTHR and beta-arrestin-2-green fluorescent protein (GFP) were found to stably colocalize during PTHR internalization, whereas after agonist removal and during receptor recycling, the colocalization slowly disappeared. Experiments using phosphorylation-deficient PTHRs and a dominant-negative form of beta-arrestin showed that beta-arrestin does not regulate the efficiency of PTHR recycling. These studies indicate that, unlike many G protein-coupled receptors, PTHR recycling does not require receptor dephosphorylation or its dissociation from beta-arrestin.
Chauvin
Stephanie
S
Endocrine Research Unit, Veterans Affairs Medical Center, University of California-San Francisco, 4150 Clement Street, San Francisco, CA 94121, USA.
Bencsik
Margaret
M
Bambino
Tom
T
Nissenson
Robert A
RA
D000818:Animals; D019390:Arrestins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D015536:Down-Regulation; D004622:Embryo, Mammalian; D004705:Endocytosis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019085:Fluorescent Antibody Technique, Direct; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics; D008387:Marine Toxins; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D019319:Okadaic Acid / Q000494:pharmacology; D009893:Opossums; D010080:Oxazoles / Q000494:pharmacology; D010281:Parathyroid Hormone / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D051381:Rats; D018016:Receptors, Parathyroid Hormone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D014162:Transfection; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
C000605254:ARRB2 protein, human; C000605257:Arrb2 protein, rat; D019390:Arrestins; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D008387:Marine Toxins; D010080:Oxazoles; D010281:Parathyroid Hormone; D018016:Receptors, Parathyroid Hormone; D011993:Recombinant Fusion Proteins; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D049452:Green Fluorescent Proteins; D019319:Okadaic Acid; C059041:calyculin A; D010749:Phosphoprotein Phosphatases