Cell-surface targeting of alpha2-adrenergic receptors -- inhibition by a transport deficient mutant through dimerization.
We previously demonstrated that the alpha2B-adrenergic receptor mutant, in which the F(x)6IL motif in the membrane-proximal carboxyl terminus were mutated to alanines (alpha2B-ARm), is deficient in export from the endoplasmic reticulum (ER). In this report, we determined if alpha2B-ARm could modulate transport from the ER to the cell surface and signaling of its wild-type counterpart. Transient expression of alpha2B-ARm in HEK293T cells markedly inhibited cell-surface expression of wild-type alpha2B-AR, as measured by radioligand binding. Subcellular localization demonstrated that alpha2B-ARm trapped alpha2B-AR in the ER. The alpha2B-AR was shown to form homodimers and heterodimers with alpha2B-ARm as measured by co-immunoprecipitation of the receptors tagged with green fluorescent protein and hemagglutinin epitopes. In addition to alpha2B-AR, the transport of alpha2A-AR and alpha2C-AR to the cell surface was also inhibited by alpha2B-ARm. Furthermore, transient expression of alpha2B-ARm significantly reduced cell-surface expression of endogenous alpha2-AR in NG108-15 and HT29 cells. Consistent with its effect on alpha2-AR cell-surface expression, alpha2B-ARm attenuated alpha2A-AR- and alpha2B-AR-mediated ERK1/2 activation. These data demonstrated that the ER-retained mutant alpha2B-ARm conferred a dominant negative effect on the cell-surface expression of wild-type alpha2-AR, which is likely mediated through heterodimerization. These data indicate a crucial role of ER export in the regulation of cell-surface targeting and signaling of G protein-coupled receptors.
Zhou
Fuguo
F
Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, 1901 Perdido St, New Orleans, LA 70112, USA.
Filipeanu
Catalin M
CM
Duvernay
Matthew T
MT
Wu
Guangyu
G
D000409:Alanine / Q000032:analysis; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D007930:Leucine / Q000032:analysis; D009154:Mutation; D010649:Phenylalanine / Q000032:analysis; D011869:Radioligand Assay; D051381:Rats; D018341:Receptors, Adrenergic, alpha-2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection
C547768:ADRA2A protein, human; C547771:ADRA2B protein, human; C547774:ADRA2C protein, human; D018341:Receptors, Adrenergic, alpha-2; D010649:Phenylalanine; D007930:Leucine; D000409:Alanine