title: Quantitative analysis of agonist-dependent parathyroid hormone receptor trafficking in whole cells using a functional green fluorescent protein conjugate.
abstract: Many G-protein coupled receptors (GPCRs) undergo ligand-dependent internalization upon activation. The parathyroid hormone (PTH) receptor undergoes endocytosis following prolonged exposure to ligand although the ultimate fate of the receptor following internalization is largely unknown. To investigate compartmentalization of the PTH receptor, we have established a stable cell line expressing a PTH receptor-green fluorescent protein (PTHR-GFP) conjugate and an algorithm to quantify PTH receptor internalization. HEK 293 cells expressing the PTHR-GFP were compared with cells expressing the wild-type PTH receptor in whole-cell binding and functional assays. 125I-PTH binding studies revealed similar Bmax and kD values in cells expressing either the PTHR-GFP or the wild-type PTH receptor. PTH-induced cAMP accumulation was similar in both cell lines suggesting that addition of the GFP to the cytoplasmic tail of the PTH receptor does not alter the ligand binding or G-protein coupling properties of the receptor. Using confocal fluorescence microscopy, we demonstrated that PTH treatment of cells expressing the PTHR-GFP conjugate produced a time-dependent redistribution of the receptor to the endosomal compartment which was blocked by pretreatment with PTH antagonist peptides. Treatment with hypertonic sucrose prevented PTH-induced receptor internalization, suggesting that the PTH receptor internalizes via a clathrin-dependent mechanism. Moreover, co-localization with internalized transferrin showed that PTHR-GFP trafficking utilized the endocytic recycling compartment. Experiments using cycloheximide to inhibit protein synthesis demonstrated that recycling of the PTHR-GFP back to the plasma membrane was complete within 1-2 h of ligand removal and was partially blocked by pretreatment with cytochalasin D, but not nocodazole. We also demonstrated that the PTH receptor, upon recycling to the plasma membrane, is capable of undergoing a second round of internalization, a finding consistent with a role for receptor recycling in functional resensitization.
authors.affiliation: Department of Drug Discovery, R.W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey, USA. bconway@prius.jnj.com
authors.forename: B R
authors.initials: BR
authors.lastname: Conway
authors.forename: L K
authors.initials: LK
authors.lastname: Minor
authors.forename: J Z
authors.initials: JZ
authors.lastname: Xu
authors.forename: M R
authors.initials: MR
authors.lastname: D'Andrea
authors.forename: R N
authors.initials: RN
authors.lastname: Ghosh
authors.forename: K T
authors.initials: KT
authors.lastname: Demarest
mesh_terms: D000465:Algorithms; D002460:Cell Line; D000242:Cyclic AMP / Q000096:biosynthesis; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects; D004705:Endocytosis; D011992:Endosomes / Q000737:chemistry; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010281:Parathyroid Hormone / Q000378:metabolism / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology; D021381:Protein Transport; D018016:Receptors, Parathyroid Hormone / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014168:Transferrin / Q000032:analysis
chemical_list: D007202:Indicators and Reagents; D008164:Luminescent Proteins; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D018016:Receptors, Parathyroid Hormone; D011993:Recombinant Fusion Proteins; D014168:Transferrin; D049452:Green Fluorescent Proteins; D015638:Cytochalasin D; C031617:parathyroid hormone (3-34); D000242:Cyclic AMP