The N Terminus of the Human α_1D-Adrenergic Receptor Prevents Cell Surface Expression

Abstract

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 (␣ 1A NT-D, ␣ 1B NT-D, ␣ 1D NT-A, and ␣ 1D NT-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 ␣ 1D NT-B-ARs were found only on the cell surface, whereas GFP-tagged ␣ 1B NT-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.

Differential Phosphorylation, Desensitization, and Internalization ofα1A−Adrenoceptors Activated by Norepinephrine and Oxymetazoline

“…HEK-293 cells transiently transfected with the p enhanced green fluorescent protein (eGFP)-N3 plasmid encoding recombinant human a1A-ARs fused at the C-terminus with enhanced green fluorescent protein [a1A/eGFP, described in Hague et al (2004) and kindly provided by Dr. Chris…”

“…Cells were transfected with 15 mg of cDNA of the mammalian expression plasmid pDT containing Nterminal sequential hexahistidine and FLAG epitope-tagged human a1A (a1A-1 splice variant), a1B, or N-terminal truncated mutant of a1D-ARs (D a1D-AR) by Lipofectamine (Invitrogen, Carlsbad, CA), and when specified, stably transfected cells were selected with Geneticin (400 mg/ml). The constructs were generously provided by Dr. K. P. Minneman and are described elsewhere (Vicentic et al, 2002;Pupo et al, 2003;Hague et al, 2004). In addition, HEK-293 cells were transfected with 20 mg of cDNA of the expression plasmid pcDNA1.1 encoding the sequence for a kinase-deficient mutant of bovine GRK2 in which lysine 220 was replaced by a methionine [C20-GRK2-K220M 3 , described in Ferguson et al (1995) (Leeb-Lundberg et al, 1987).…”

α-Dystrobrevin-1 recruits α-catulin to the α 1D -adrenergic receptor/dystrophin-associated protein complex signalosome

“…In agreement with previous studies, α 1D -AR displays low levels of functional activity with rate-limiting concentrations of syntrophins in cells (Fig. 4A) (9,26). To circumvent this technical issue, we used an α 1D -AR-syntrophin fusion protein separated by a six-glycine linker (10).…”

“…A member of the adrenergic family (α 1 , α 2 , β), α 1D -ARs are ubiquitously expressed on blood vessels and are responsible for increasing blood pressure during exercise, injury, stress, or cardiovascular disease (6). α 1D -AR knockout mice are hypotensive and resistant to high salt diet-induced hypertension (7,8), yet this GPCR has been largely ignored over the past 20 y because after transfection into cell culture α 1D -ARs are sequestered in the endoplasmic reticulum (9,10). Clinical interest in the α 1D -AR as a drug target has recently increased with the discoveries that α 1D -ARs are the predominant subtype expressed in epicardial coronary arteries (11) and that α 1D -AR prostate expression increases in patients with benign prostatic hypertrophy (12).…”

α1D-Adrenergic receptor insensitivity is associated with alterations in its expression and distribution in cultured vascular myocytes

“…Recently, several lines of evidence have implicated some critical factors for modulating α 1D -AR functional expression at the cell membrane. For example, the truncation of 79 amino acids from the receptors' N-termini results in translocation of the α 1D -ARs from intracellular compartments onto the plasma membrane, and a three-to four-fold increase in IP 3 formation due to norepinephrine stimulation [41] . Additionally, dystrophin proteins, a type of intracellular anchor protein, have been identified as essential elements for α 1D -AR but not for α 1A -or α 1B -AR functional expression for both in vitro and in vivo situations [42] .…”