The N terminus of the human alpha1D-adrenergic receptor prevents cell surface expression.
We previously reported that truncation of the N-terminal 79 amino acids of alpha(1D)-adrenoceptors (Delta(1-79)alpha(1D)-ARs) greatly increases binding site density. In this study, we determined whether this effect was associated with changes in alpha(1D)-AR subcellular localization. Confocal imaging of green fluorescent protein (GFP)-tagged receptors and sucrose density gradient fractionation suggested that full-length alpha(1D)-ARs were found primarily in intracellular compartments, whereas Delta(1-79)alpha(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 alpha(1)-ARs containing the body of one subtype and the N terminus of another (alpha(1A)NT-D, alpha(1B)NT-D, alpha(1D)NT-A, and alpha(1D)NT-B). When expressed in human embryonic kidney 293 cells, radioligand binding revealed that binding densities of alpha(1A)-or alpha(1B)-ARs containing the alpha(1D)-N terminus decreased by 86 to 93%, whereas substitution of alpha(1A)- or alpha(1B)-N termini increased alpha(1D)-AR binding site density by 2- to 3-fold. Confocal microscopy showed that GFP-tagged alpha(1D)NT-B-ARs were found only on the cell surface, whereas GFP-tagged alpha(1B)NT-D-ARs were completely intracellular. Radioligand binding and confocal imaging of GFP-tagged alpha(1D)- and Delta(1-79)alpha(1D)-ARs expressed in rat aortic smooth muscle cells produced similar results, suggesting these effects are generalizable to cell types that endogenously express alpha(1D)-ARs. These findings demonstrate that the N-terminal region of alpha(1D)-ARs contain a transplantable signal that is critical for regulating formation of functional bindings, through regulating cellular localization.
Hague
Chris
C
Department of Pharmacology, Emory University Medical School, Rollins Research Building, 1510 Clifton Road, Atlanta, GA 30322, USA. chague@emory.edu
Chen
Zhongjian
Z
Pupo
Andre S
AS
Schulte
Nancy A
NA
Toews
Myron L
ML
Minneman
Kenneth P
KP
D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D008164:Luminescent Proteins; D009638:Norepinephrine / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000502:physiology*; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay; D018340:Receptors, Adrenergic, alpha-1 / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection
C547719:ADRA1D protein, human; C547721:Adra1d protein, rat; D007295:Inositol Phosphates; D008164:Luminescent Proteins; D021382:Protein Sorting Signals; D018340:Receptors, Adrenergic, alpha-1; D049452:Green Fluorescent Proteins; D009638:Norepinephrine