Quantitative analysis of agonist-dependent parathyroid hormone receptor trafficking in whole cells using a functional green fluorescent protein conjugate

Many G-protein coupled receptors (GPCRs) undergo ligand-dependent internali zation upon activation. The parathyroid hormone (PTH) receptor undergoes en docytosis following prolonged exposure to ligand although the ultimate fate of the receptor following internalization is largely unknown. To investiga te compartmentalization of the PTH receptor, we have established a stable c ell line expressing a PTH receptor-green fluorescent protein (PTHR-GFP) con jugate and an algorithm to quantify PTH receptor internalization. HEK 293 c ells expressing the PTHR-GFP were compared with cells expressing the wild-t ype PTH receptor in whole-cell binding and functional assays. I-125-PTH bin ding studies revealed similar B-max and kD values in cells expressing eithe r the PTHR-GFP or the wildtype PTH receptor. PTH-Induced cAMP accumulation was similar in both cell lines suggesting that addition of the GFP to the c ytoplasmic tail of the PTH receptor does not alter the ligand binding or G- protein coupling properties of the receptor. Using confocal fluorescence mi croscopy, we demonstrated that PTH treatment of cells expressing the PTHR-G FP conjugate produced a time-dependent redistribution of the receptor to th e endosomal compartment which was blocked by pretreatment with PTH antagoni st peptides. Treatment with hypertonic sucrose prevented PTH-induced recept or internalization, suggesting that the PTH receptor internalizes via a cla thrin-dependent mechanism. Moreover, co-localization with internalized tran sferrin showed that PTHR-GFP trafficking utilized the endocytic recycling c ompartment. Experiments using cycloheximide to inhibit protein synthesis de monstrated that recycling of the PTHR-GFP back to the plasma membrane was c omplete within 1-2 h of ligand removal and was partially blocked by pretrea tment with cytochalasin D, but not nocodazole. We also demonstrated that th e PTH receptor, upon recycling to the plasma membrane, is capable of underg oing a second round of internalization, a finding consistent with a role fo r receptor recycling in functional resensitization. (C) 2001 Wiley-Liss, In c.