Cellular distribution of constitutively active mutant parathyroid hormone (PTH)/PTH-related protein receptors and regulation of cyclic adenosine 3 ',5 '-monophosphate signaling by beta-arrestin2

PTH promotes endocytosis of human PTH receptor 1 (PTH1Rc) by activating pro tein kinase C and recruiting beta -arrestin2. We examined the role of beta -arrestin2 in regulating the cellular distribution and cAMP signaling of tw o constitutively active PTH1Rc mutants, H223R and T410P. Overexpression of a beta -arrestin2-green fluorescent protein (GFP) conjugate in COS-7 cells inhibited constitutive cAMP accumulation by H223R and T410P in a dose-depen dent manner, as well as the response to PTH of both mutant and wild-type PT H1Rcs. The cellular distribution of PTH1Rc-GFP conjugates, fluorescent liga nds, and beta arrestin2-GFP was analyzed by fluorescence microscopy in HEK- 293T cells. In cells expressing either receptor mutant, a ligand-independen t mobilization of beta -arrestin2 to the cell membrane was observed. In the absence of ligand, H223R and wild-type PTH1Rcs were mainly localized on th e cell membrane, whereas intracellular trafficking of T410P was also observ ed. While agonists promoted beta -arrestin2-mediated endocytosis of both PT H1Rc mutants, antagonists were rapidly internalized only with T410P. The pr otein kinases inhibitor, staurosporine, significantly decreased internaliza tion of ligand-PTH1Rc mutant complexes, although the recruitment of beta -a rrestin2 to the cell membrane was unaffected. Moreover, in cells expressing a truncated wild-type PTH1Rc lacking the C-terminal cytoplasmic domain, ag onists stimulated translocation of beta -arrestin2 to the cell membrane fol lowed by ligand-receptor complex internalization without associated beta -a rrestin2. In conclusion, cAMP signaling by constitutively active mutant and wild-type PTH1Rcs is inhibited by a receptor interaction with beta -arrest in2 on the cell membrane, possibly leading to uncoupling from G(s)alpha. Th is phenomenon is independent from protein kinases activity and the receptor C-terminal cytoplasmic domain. In addition, there are differences in the c ellular localization and internalization features of constitutively active PTH1Rc mutants H223R and T410P.