N-terminal truncation of human alpha1D-adrenoceptors increases expression of binding sites but not protein

Eur J Pharmacol. 2003 Feb 21;462(1-3):1-8. doi: 10.1016/s0014-2999(03)01292-5.

Abstract

The role of the N-terminus of human alpha(1D)-adrenoceptors was examined by deleting the first 79 amino acids (Delta(1-79)) and epitope-tagging to facilitate immunoprecipitation and detection. Following transfection into HEK293 cells, 6- to 13-fold increases in the density of specific [125I]BE 2254 binding sites were observed for both tagged and untagged Delta(1-79)alpha(1D)- compared to full-length alpha(1D)-adrenoceptors, while agonist and antagonist affinities remained unchanged. In contrast, immunoprecipitation of tagged receptors showed that full-length alpha(1D)-adrenoceptor protein was at least twice as abundant as Delta(1-79)alpha(1D)-adrenoceptor protein. Photoaffinity labelling with [125I]arylazidoprazosin showed much more intense labelling of tagged Delta(1-79)alpha(1D)- than of full-length alpha(1D)-adrenoceptors. Substantial N-linked glycosylation of tagged Delta(1-79)alpha(1D)-adrenoceptors was observed, although full-length alpha(1D)-adrenoceptors contain two consensus glycosylation sites but are not glycosylated. These results suggest that N-terminal truncation of alpha(1D)-adrenoceptors enhances processing of a binding competent form in HEK293 cells; and show a clear dissociation between abundance of receptor protein and density of receptor binding sites.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Azides / metabolism
  • Binding Sites
  • Binding, Competitive
  • Calcium / metabolism
  • Cell Line
  • Glycosylation
  • Humans
  • Iodine Radioisotopes
  • Kinetics
  • Molecular Sequence Data
  • Norepinephrine / pharmacology
  • Oligopeptides
  • Peptides / genetics
  • Phenethylamines / metabolism
  • Photoaffinity Labels / metabolism
  • Prazosin / analogs & derivatives*
  • Prazosin / metabolism
  • Precipitin Tests
  • Radioligand Assay
  • Receptors, Adrenergic, alpha-1 / drug effects
  • Receptors, Adrenergic, alpha-1 / genetics
  • Receptors, Adrenergic, alpha-1 / metabolism*
  • Sequence Deletion*
  • Tetralones*
  • Transfection
  • Uridine Triphosphate / pharmacology

Substances

  • ADRA1D protein, human
  • Azides
  • Iodine Radioisotopes
  • Oligopeptides
  • Peptides
  • Phenethylamines
  • Photoaffinity Labels
  • Receptors, Adrenergic, alpha-1
  • Tetralones
  • BE 2254
  • azidoprazosin
  • FLAG peptide
  • Calcium
  • Uridine Triphosphate
  • Norepinephrine
  • Prazosin