Cellular Distribution of Constitutively Active Mutant Parathyroid Hormone (PTH)/PTH-Related Protein Receptors and Regulation of Cyclic Adenosine 3′,5′-Monophosphate Signaling by β-Arrestin2

PTH promotes endocytosis of human PTH receptor 1 (PTH1Rc) by activating protein kinase C and recruitingβ -arrestin2. We examined the role of β-arrestin2 in regulating the cellular distribution and cAMP signaling of two constitutively active PTH1Rc mutants, H223R and T410P. Overexpression of aβ -arrestin2-green fluorescent protein (GFP) conjugate in COS-7 cells inhibited constitutive cAMP accumulation by H223R and T410P in a dose-dependent manner, as well as the response to PTH of both mutant and wild-type PTH1Rcs. The cellular distribution of PTH1Rc-GFP conjugates, fluorescent ligands, and βarrestin2-GFP was analyzed by fluorescence microscopy in HEK-293T cells. In cells expressing either receptor mutant, a ligand-independent mobilization ofβ -arrestin2 to the cell membrane was observed. In the absence of ligand, H223R and wild-type PTH1Rcs were mainly localized on the cell membrane, whereas intracellular trafficking of T410P was also observed. While agonists promoted β-arrestin2-mediated endocytosis of b...