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

Mol Endocrinol. 2001 Jan;15(1):149-63. doi: 10.1210/mend.15.1.0587.


PTH promotes endocytosis of human PTH receptor 1 (PTH1Rc) by activating protein kinase C and recruiting beta-arrestin2. We examined the role of beta-arrestin2 in regulating the cellular distribution and cAMP signaling of two 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-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 ssarrestin2-GFP was analyzed by fluorescence microscopy in HEK-293T cells. In cells expressing either receptor mutant, a ligand-independent mobilization of beta-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 beta-arrestin2-mediated endocytosis of bot PTH1Rc mutants, antagonists were rapidly internalized only with T410P. The protein kinases inhibitor, staurosporine, significantly decreased internalization of ligand-PTH1Rc mutant complexes, although the recruitment of beta-arrestin2 to the cell membrane was unaffected. Moreover, in cells expressing a truncated wild-type PTH1Rc lacking the C-terminal cytoplasmic domain, agonists stimulated translocation of beta-arrestin2 to the cell membrane followed by ligand-receptor complex internalization without associated beta-arrestin2. In conclusion, cAMP signaling by constitutively active mutant and wild-type PTH1Rcs is inhibited by a receptor interaction with beta-arrestin2 on the cell membrane, possibly leading to uncoupling from G(s)alpha. This phenomenon is independent from protein kinases activity and the receptor C-terminal cytoplasmic domain. In addition, there are differences in the cellular localization and internalization features of constitutively active PTH1Rc mutants H223R and T410P.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Arrestins / genetics
  • Arrestins / metabolism
  • Arrestins / pharmacology*
  • COS Cells
  • Cell Membrane / chemistry
  • Cell Membrane / metabolism
  • Cyclic AMP / metabolism*
  • Endocytosis / drug effects
  • Enzyme Inhibitors / pharmacology
  • Green Fluorescent Proteins
  • Humans
  • Luminescent Proteins / genetics
  • Microscopy, Fluorescence
  • Mutation*
  • Protein Kinase Inhibitors
  • Receptor, Parathyroid Hormone, Type 1
  • Receptors, Parathyroid Hormone / analysis*
  • Receptors, Parathyroid Hormone / genetics
  • Receptors, Parathyroid Hormone / metabolism
  • Recombinant Fusion Proteins / analysis
  • Recombinant Fusion Proteins / metabolism
  • Signal Transduction / drug effects*
  • Staurosporine / pharmacology
  • Transfection
  • beta-Arrestins


  • Arrestins
  • Enzyme Inhibitors
  • Luminescent Proteins
  • Protein Kinase Inhibitors
  • Receptor, Parathyroid Hormone, Type 1
  • Receptors, Parathyroid Hormone
  • Recombinant Fusion Proteins
  • beta-Arrestins
  • Green Fluorescent Proteins
  • Cyclic AMP
  • Staurosporine