N-terminal truncation of human alpha1D-adrenoceptors increases expression of binding sites but not protein.
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.
Pupo
Andre S
AS
Department of Pharmacology, 5017 Rollins Research Center, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA.
Uberti
Michelle A
MA
Minneman
Kenneth P
KP
D000595:Amino Acid Sequence; D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D006031:Glycosylation; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D008969:Molecular Sequence Data; D009638:Norepinephrine / Q000494:pharmacology; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics; D010627:Phenethylamines / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism; D011224:Prazosin / Q000031:analogs & derivatives* / Q000378:metabolism; D011233:Precipitin Tests; D011869:Radioligand Assay; D018340:Receptors, Adrenergic, alpha-1 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D044024:Tetralones; D014162:Transfection; D014544:Uridine Triphosphate / Q000494:pharmacology
C547719:ADRA1D protein, human; D001386:Azides; D007457:Iodine Radioisotopes; D009842:Oligopeptides; D010455:Peptides; D010627:Phenethylamines; D019920:Photoaffinity Labels; D018340:Receptors, Adrenergic, alpha-1; D044024:Tetralones; C010652:BE 2254; C041552:azidoprazosin; C085706:FLAG peptide; D002118:Calcium; D014544:Uridine Triphosphate; D009638:Norepinephrine; D011224:Prazosin