The zeta isoform of 14-3-3 proteins interacts with the third intracellular loop of different alpha2-adrenergic receptor subtypes.
The alpha2-adrenergic receptors (alpha2ARs) are localized to and function on the basolateral surface in polarized renal epithelial cells via a mechanism involving the third cytoplasmic loop. To identify proteins that may contribute to this retention, [35S]Met-labeled Gen10 fusion proteins with the 3i loops of the alpha2AAR (Val217-Ala377), alpha2BAR (Lys210-Trp354), and alpha2CAR (Arg248-Val363) were used as ligands in gel overlay assays. A protein doublet of approximately 30 kDa in Madin-Darby canine kidney cells or pig brain cytosol (alpha2B >/= alpha2C>> alpha2A) was identified. The interacting protein was purified by sequential DEAE and size exclusion chromatography, and subsequent microsequencing revealed that they are the zeta isoform of 14-3-3 proteins. [35S]Met-14-3-3zeta binds to all three native alpha2AR subtypes, assessed using a solid phase binding assay (alpha2A>/=alpha2B> alpha2C), and this binding depends on the presence of the 3i loops. Attenuation of the alpha2AR-14-3-3 interactions in the presence of a phosphorylated Raf-1 peptide corresponding to its 14-3-3 interacting domain (residues 251-266), but not by its non-phosphorylated counterpart, provides evidence for the functional specificity of these interactions and suggests one potential interface for the alpha2AR and 14-3-3 interactions. These studies represent the first evidence for G protein-coupled receptor interactions with 14-3-3 proteins and may provide a mechanism for receptor localization and/or coordination of signal transduction.
Prezeau
L
L
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600, USA.
Richman
J G
JG
Edwards
S W
SW
Limbird
L E
LE
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004285:Dogs; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D018341:Receptors, Adrenergic, alpha-2 / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013552:Swine; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D011506:Proteins; D018341:Receptors, Adrenergic, alpha-2; D014446:Tyrosine 3-Monooxygenase