The N Terminus of the Human α1D-Adrenergic Receptor Prevents Cell Surface Expression

• Published in: The Journal of pharmacology and experimental therapeutics 2004-04, Vol.309 (1), p.388
• Main Authors: Chris Hague, Zhongjian Chen, Andre S. Pupo, Nancy A. Schulte, Myron L. Toews, Kenneth P. Minneman
• Format: Article
• Language: English
• ISSN: 0022-3565; 1521-0103
• DOI: 10.1124/jpet.103.060509

We previously reported that truncation of the N-terminal 79 amino acids of α 1D -adrenoceptors (Δ 1-79 α 1D -ARs) greatly increases binding site density. In this study, we determined whether this effect was associated with changes in α 1D -AR subcellular localization. Confocal imaging of green fluorescent protein (GFP)-tagged receptors and sucrose density gradient fractionation suggested that full-length α 1D -ARs were found primarily in intracellular compartments, whereas Δ 1-79 α 1D -ARs were translocated to the plasma membrane. This resulted in a 3- to 4-fold increase in intrinsic activity for stimulation of inositol phosphate formation by norepinephrine. We determined whether this effect was transplantable by creating N-terminal chimeras of α 1 -ARs containing the body of one subtype and the N terminus of another (α 1A NT-D, α 1B NT-D, α 1D NT-A, and α 1D NT-B). When expressed in human embryonic kidney 293 cells, radioligand binding revealed that binding densities of α 1A -or α 1B -ARs containing the α 1D -N terminus decreased by 86 to 93%, whereas substitution of α 1A - or α 1B -N termini increased α 1D -AR binding site density by 2- to 3-fold. Confocal microscopy showed that GFP-tagged α 1D NT-B-ARs were found only on the cell surface, whereas GFP-tagged α 1B NT-D-ARs were completely intracellular. Radioligand binding and confocal imaging of GFP-tagged α 1D - and Δ 1-79 α 1D -ARs expressed in rat aortic smooth muscle cells produced similar results, suggesting these effects are generalizable to cell types that endogenously express α 1D -ARs. These findings demonstrate that the N-terminal region of α 1D -ARs contain a transplantable signal that is critical for regulating formation of functional bindings, through regulating cellular localization.