Addition of a signal peptide sequence to the alpha1D-adrenoceptor gene increases the density of receptors, as determined by [3H]-prazosin binding in the membranes.
1. Both in mammalian tissues and in transfected cells, only low levels of alpha1D-adrenoceptors are detected in radioligand binding studies. It has been implicated that the comparatively long N-terminal tail of the alpha1D-adrenoceptor is responsible for the inefficient surface expression of the receptor. 2. In the present study, we created gene constructs for six N-terminally truncated variants of the human alpha1D-adrenoceptor. These constructs were used to transfect Neuro2A cells. We show that the density of alpha1D-adrenoceptors, observed by [3H]-prazosin binding, gradually increased with longer truncations of the N-terminus. This seems to indicate that the long N-terminal tail nonspecifically interferes with receptor translocation to the plasma membrane. 3. The addition of a 16 amino acids long signal peptide to the N-terminus of the wild-type alpha1D-adrenoceptor increased the density of receptor binding sites 10-fold in Neuro2A and COS-7 cells. This indicates that, after the addition of a signal peptide, the long N-terminal tail of the alpha1D-adrenoceptor does not interfere with proper translocation of the receptor to the plasma membrane. This, in turn, indicates that the N-terminal tail of the wild-type alpha1D-adrenoceptor, merely by its long length, hinders the first transmembrane helix of the receptor from being a signal anchor. 4. Neither the wild-type alpha1D-adrenoceptor (for which the expression level of [3H]-prazosin binding sites is low) nor the truncated alpha1D-adrenoceptor variant (for which the expression level of [3H]-prazosin binding sites is high) showed any constitutive activity in stimulating inositol phosphate accumulation. This indicates that the low expression level of [3H]-prazosin binding sites, after transfection with the wild-type alpha1D-adrenoceptor, is not caused by constitutive activity of the receptor and subsequent receptor downregulation.
Petrovska
Ramona
R
Department of Pharmaceutical Biosciences, Unit of Pharmacology, Uppsala University, Box 591, BMC, Uppsala SE-751 24, Sweden.
Kapa
Ivo
I
Klovins
Janis
J
Schiöth
Helgi B
HB
Uhlén
Staffan
S
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism; D002522:Chlorocebus aethiops; D007093:Imidazoles / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D051379:Mice; D010656:Phenylephrine / Q000494:pharmacology; D011224:Prazosin / Q000494:pharmacology*; D011487:Protein Conformation; D021382:Protein Sorting Signals; D018340:Receptors, Adrenergic, alpha-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014316:Tritium
C547713:ADRA1A protein, human; C547716:ADRA1B protein, human; C547719:ADRA1D protein, human; C547714:Adra1a protein, mouse; C547717:Adra1b protein, mouse; C547720:Adra1d protein, mouse; D000316:Adrenergic alpha-Agonists; D000317:Adrenergic alpha-Antagonists; D007093:Imidazoles; D007295:Inositol Phosphates; D021382:Protein Sorting Signals; D018340:Receptors, Adrenergic, alpha-1; D014316:Tritium; D010656:Phenylephrine; C014282:cirazoline; D011224:Prazosin