Cell surface expression of alpha1D-adrenergic receptors is controlled by heterodimerization with alpha1B-adrenergic receptors

J Biol Chem. 2004 Apr 9;279(15):15541-9. doi: 10.1074/jbc.M314014200. Epub 2004 Jan 21.

Abstract

alpha(1)-Adrenergic receptors (ARs) belong to the large Class I G protein-coupled receptor superfamily and comprise three subtypes (alpha(1A), alpha(1B), and alpha(1D)). Previous work with heterologously expressed C-terminal green fluorescent protein (GFP)-tagged alpha(1)-ARs showed that alpha(1A)- and alpha(1B)-ARs localize to the plasma membrane, whereas alpha(1D)-ARs accumulate intracellularly. We recently showed that alpha(1D)- and alpha(1B)-ARs form heterodimers, whereas alpha(1D)- and alpha(1A)-ARs do not. Here, we examined the role of heterodimerization in regulating alpha(1D)-AR localization using both confocal imaging of GFP- or CFP-tagged alpha(1)-ARs and a luminometer-based surface expression assay in HEK293 cells. Co-expression with alpha(1B)-ARs caused alpha(1D)-ARs to quantitatively translocate to the cell surface, but co-expression with alpha(1A)-ARs did not. Truncation of the alpha(1B)-AR extracellular N terminus or intracellular C terminus had no effect on surface expression of alpha(1D)-ARs, suggesting primary involvement of the hydrophobic core. Co-transfection with an uncoupled mutant alpha(1B)-AR (Delta12alpha(1B)) increased both alpha(1D)-AR surface expression and coupling to norepinephrine-stimulated Ca(2+) mobilization. Finally, GFP-tagged alpha(1D)-ARs were not detected on the cell surface when expressed in rat aortic smooth muscle cells that express no endogenous ARs, but were almost exclusively localized on the surface when expressed in DDT(1)MF-2 cells, which express endogenous alpha(1B)-ARs. These studies demonstrate that alpha(1B)/alpha(1D)-AR heterodimerization controls surface expression and functional coupling of alpha(1D)-ARs, the N- and C-terminal domains are not involved in this interaction, and that alpha(1B)-AR G protein coupling is not required. These observations may be relevant to many other Class I G protein-coupled receptors, where the functional consequences of heterodimerization are still poorly understood.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Calcium / metabolism
  • Cell Line
  • Cell Membrane / metabolism*
  • Cricetinae
  • Dimerization
  • Green Fluorescent Proteins
  • Humans
  • Inositol Phosphates / chemistry
  • Luminescent Proteins / metabolism
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • Molecular Sequence Data
  • Muscle, Smooth / cytology
  • Protein Structure, Tertiary
  • Protein Transport
  • Rats
  • Receptors, Adrenergic, alpha-1 / biosynthesis*
  • Recombinant Fusion Proteins / metabolism
  • Transfection

Substances

  • ADRA1B protein, human
  • ADRA1D protein, human
  • Adra1b protein, rat
  • Adra1d protein, rat
  • Inositol Phosphates
  • Luminescent Proteins
  • Receptors, Adrenergic, alpha-1
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins
  • Calcium