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

Publisher Website
Google Scholar
Abstract
We previously reported that truncation of the N-terminal 79 amino acids of α1D-adrenoceptors (Δ1-79α1D-ARs) greatly increases binding site density. In this study, we determined whether this effect was associated with changes in α1D-AR subcellular localization. Confocal imaging of green fluorescent protein (GFP)-tagged receptors and sucrose density gradient fractionation suggested that full-length α1D-ARs were found primarily in intracellular compartments, whereas Δ1-79α1D-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 α1-ARs containing the body of one subtype and the N terminus of another (α1ANT-D, α1BNT-D, α1DNT-A, and α1DNT-B). When expressed in human embryonic kidney 293 cells, radioligand binding revealed that binding densities of α1A-or α1B-ARs containing the α1D-N terminus decreased by 86 to 93%, whereas substitution of α1A- or α1B-N termini increased α1D-AR binding site density by 2- to 3-fold. Confocal microscopy showed that GFP-tagged α1DNT-B-ARs were found only on the cell surface, whereas GFP-tagged α1BNT-D-ARs were completely intracellular. Radioligand binding and confocal imaging of GFP-tagged α1D- and Δ1-79α1D-ARs expressed in rat aortic smooth muscle cells produced similar results, suggesting these effects are generalizable to cell types that endogenously express α1D-ARs. These findings demonstrate that the N-terminal region of α1D-ARs contain a transplantable signal that is critical for regulating formation of functional bindings, through regulating cellular localization.