Mechanisms regulating the cell surface residence time of the alpha 2A-adrenergic receptor

Biochemistry. 2000 Feb 1;39(4):693-700. doi: 10.1021/bi9920275.

Abstract

Despite considerable insights concerning the mechanisms regulating short-term agonist-mediated G protein-coupled receptor (GPCR) internalization, little is known about the mechanisms regulating GPCR surface residence over long periods of time. Herein, we experimentally evaluated mechanisms regulating the surface t(1/2) of various alpha(2A)-adrenergic receptor (alpha(2A)AR) structures. The Delta 3i alpha(2A)AR (lacking the third intracellular loop), D79N alpha(2A)AR (impaired G protein coupling), and CAM alpha(2A)AR (enhanced G protein coupling) all exhibited a cell surface alpha(2A)AR turnover in Chinese hamster ovary cells that was faster than that of the wild type (WT). Cell surface receptor turnover could be slowed with ligand occupancy of D79N alpha(2A)AR (agonist or antagonist) and CAM alpha(2A)AR (antagonist only) but not the Delta 3i- or WT alpha(2A)AR. This selective ligand-induced surface stabilization was paralleled by a dramatic ligand-dependent receptor density upregulation for D79N- and CAM alpha(2A)AR structures. Receptors which exhibited surface turnover and density that could be modulated by ligand (D79N and CAM) also demonstrated structural instability, measured by a loss of radioligand binding capacity in detergent solution over time without parallel changes in receptor protein content. In contrast, the shorter surface t(1/2) of the Delta 3i alpha(2A)AR, whose cell surface t(1/2) and steady state density were not altered by ligand occupancy, occurred in the context of a structurally stable receptor in detergent solution. These results demonstrate that changes in receptor structure which alter receptor-G protein coupling (either an increase or decrease) are paralleled by structural instability and ligand-induced surface stabilization. These studies also provide criteria for assessing the structural instability of the alpha(2A)AR that can likely be generalized to all GPCRs.

Publication types

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

MeSH terms

  • Adrenergic alpha-2 Receptor Agonists
  • Adrenergic alpha-2 Receptor Antagonists
  • Animals
  • Asparagine / genetics
  • Aspartic Acid / genetics
  • Binding Sites / genetics
  • CHO Cells
  • Cell Membrane / metabolism
  • Cricetinae
  • GTP-Binding Proteins / metabolism
  • Half-Life
  • Kinetics
  • Ligands
  • Receptors, Adrenergic, alpha-2 / genetics
  • Receptors, Adrenergic, alpha-2 / metabolism*
  • Time Factors
  • Transfection
  • Up-Regulation / genetics

Substances

  • Adrenergic alpha-2 Receptor Agonists
  • Adrenergic alpha-2 Receptor Antagonists
  • Ligands
  • Receptors, Adrenergic, alpha-2
  • Aspartic Acid
  • Asparagine
  • GTP-Binding Proteins