Abstract
alpha(1)-Adrenergic receptors (ARs) belong to the large Class I G protein-coupled receptor superfamily and comprise three subtypes (alpha(1A), alpha(1B), and alpha(1D)). Previous work with heterologously expressed C-terminal green fluorescent protein (GFP)-tagged alpha(1)-ARs showed that alpha(1A)- and alpha(1B)-ARs localize to the plasma membrane, whereas alpha(1D)-ARs accumulate intracellularly. We recently showed that alpha(1D)- and alpha(1B)-ARs form heterodimers, whereas alpha(1D)- and alpha(1A)-ARs do not. Here, we examined the role of heterodimerization in regulating alpha(1D)-AR localization using both confocal imaging of GFP- or CFP-tagged alpha(1)-ARs and a luminometer-based surface expression assay in HEK293 cells. Co-expression with alpha(1B)-ARs caused alpha(1D)-ARs to quantitatively translocate to the cell surface, but co-expression with alpha(1A)-ARs did not. Truncation of the alpha(1B)-AR extracellular N terminus or intracellular C terminus had no effect on surface expression of alpha(1D)-ARs, suggesting primary involvement of the hydrophobic core. Co-transfection with an uncoupled mutant alpha(1B)-AR (Delta12alpha(1B)) increased both alpha(1D)-AR surface expression and coupling to norepinephrine-stimulated Ca(2+) mobilization. Finally, GFP-tagged alpha(1D)-ARs were not detected on the cell surface when expressed in rat aortic smooth muscle cells that express no endogenous ARs, but were almost exclusively localized on the surface when expressed in DDT(1)MF-2 cells, which express endogenous alpha(1B)-ARs. These studies demonstrate that alpha(1B)/alpha(1D)-AR heterodimerization controls surface expression and functional coupling of alpha(1D)-ARs, the N- and C-terminal domains are not involved in this interaction, and that alpha(1B)-AR G protein coupling is not required. These observations may be relevant to many other Class I G protein-coupled receptors, where the functional consequences of heterodimerization are still poorly understood.
Highlights
Materials—Materials were obtained from the following sources: cDNAs for the wild type human ␣1A-Adrenergic receptors (ARs) [40], and human ␣1A, ␣1B, and ␣1D-AR C-terminally tagged green fluorescent protein (GFP) constructs in pEGFP-N3 [12, 13] were generously provided by Dr Gozoh Tsujimoto (National Children’s Hospital, Tokyo, Japan), human ␣1B-AR cDNA [41] was a gift from Dr Dianne Perez (Cleveland Clinic, Cleveland, OH), human ␣1D-AR cDNA was cloned in our laboratory [42]; pECFP-N1 vector was a gift from Dr John Hepler (Emory University, Atlanta, GA); hamster ␣1B-AR and ⌬12␣1B-AR in pCMV were a gift from Dr Myron Toews (University of Nebraska Medical Center, Omaha, NE); rat aortic smooth muscle (RASM) cells were a gift from Dr T
Cellular Localization of ␣1-AR Subtypes—To provide an overview of the subcellular localization patterns of individually expressed ␣1-AR subtypes, ␣1-ARs tagged at the C terminus with either GFP or CFP were transiently transfected into HEK293 cells, fixed on coverslips, and examined using confocal microscopy
In comparison to transient expression of ␣1D-ARs alone, co-expression of ␣1B- with ␣1DARs resulted in a significant increase (6.7-fold) in ␣1D-AR cell surface expression, which is strikingly similar to the -fold increase (6.6-fold) in ␣1D-AR cell surface expression observed with cell counts (Table I)
Summary
Materials—Materials were obtained from the following sources: cDNAs for the wild type human ␣1A-AR [40], and human ␣1A-, ␣1B-, and ␣1D-AR C-terminally tagged GFP constructs in pEGFP-N3 [12, 13] were generously provided by Dr Gozoh Tsujimoto (National Children’s Hospital, Tokyo, Japan), human ␣1B-AR cDNA [41] was a gift from Dr Dianne Perez (Cleveland Clinic, Cleveland, OH), human ␣1D-AR cDNA was cloned in our laboratory [42]; pECFP-N1 vector was a gift from Dr John Hepler (Emory University, Atlanta, GA); hamster ␣1B-AR and ⌬12␣1B-AR in pCMV were a gift from Dr Myron Toews (University of Nebraska Medical Center, Omaha, NE); rat aortic smooth muscle (RASM) cells were a gift from Dr T. Laser Confocal Microscopy—Cells transiently transfected with HA-, CFP-, or GFP-tagged constructs were grown on sterile coverslips, fixed for 30 min with 2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, and rinsed 3ϫ with PBS containing 0.5% normal horse serum (PBSϩ). Confluent 150-mm plates of transiently transfected HEK293 cells were washed with biological salt solution (BSS) (in mM: NaCl, 130; KCl, 5; MgCl2, 1; CaCl2, 1.5; HEPES, 20; glucose, 10; with 0.1% BSA), gently detached using trypsin in Ca2ϩ-free Hanks’ solution, and centrifuged for 2 min at 1000 ϫ g at 4 °C. Transfected HEK293 cells were prelabeled with myo-[3H]inositol for 48 h, and production of [3H]InsP was determined by modification of a protocol described previously [45]. Mean values were compared using the unpaired t test, with a p value less 0.05 considered significant
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