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CELLULAR AND MOLECULAR

The N Terminus of the Human _1D-Adrenergic Receptor Prevents Cell Surface Expression

Department of Pharmacology, Emory University Medical School, Atlanta, Georgia (C.H., Z.C., K.P.M.); Department of Pharmacology, Instituto de Biociencias, UNESP, Botucatu, Sao Paulo, Brazil (A.S.P., N.A.S.); and Department of Pharmacology, University of Nebraska Medical Center, Omaha, Nebraska (M.L.T.)

We previously reported that truncation of the N-terminal 79 amino acids of _1D-adrenoceptors (^1-79_1D-ARs) greatly increases binding site density. In this study, we determined whether this effect was associated with changes in _1D-AR subcellular localization. Confocal imaging of green fluorescent protein (GFP)-tagged receptors and sucrose density gradient fractionation suggested that full-length _1D-ARs were found primarily in intracellular compartments, whereas ^1-79_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 ₁-ARs containing the body of one subtype and the N terminus of another (_1ANT-D, _1BNT-D, _1DNT-A, and _1DNT-B). When expressed in human embryonic kidney 293 cells, radioligand binding revealed that binding densities of _1A-or _1B-ARs containing the _1D-N terminus decreased by 86 to 93%, whereas substitution of _1A- or _1B-N termini increased _1D-AR binding site density by 2- to 3-fold. Confocal microscopy showed that GFP-tagged _1DNT-B-ARs were found only on the cell surface, whereas GFP-tagged _1BNT-D-ARs were completely intracellular. Radioligand binding and confocal imaging of GFP-tagged _1D- and ^1-79_1D-ARs expressed in rat aortic smooth muscle cells produced similar results, suggesting these effects are generalizable to cell types that endogenously express _1D-ARs. These findings demonstrate that the N-terminal region of _1D-ARs contain a transplantable signal that is critical for regulating formation of functional bindings, through regulating cellular localization.

Address correspondence to: Dr. Chris Hague, Department of Pharmacology, Rollins Research Bldg., 1510 Clifton Rd., Emory University, Atlanta, GA 30322. E-mail: chague{at}emory.edu

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