Abstract
The third intracellular (3i) loops of the alpha 2A- and alpha 2B-adrenergic receptor (AR) subtypes are critical for retention of these receptors at the basolateral surface of polarized Madin-Darby canine kidney (MDCKII) cells at steady state. The third intracellular loops of the alpha 2A, alpha 2B, and alpha 2C-AR subtypes interact with spinophilin, a multidomain protein that, like the three alpha 2-AR subtypes, is enriched at the basolateral surface of MDCKII cells. The present studies provide evidence that alpha 2-AR interaction with spinophilin contributes to cell surface stabilization of the receptor. We exploited the unique targeting profile of the alpha 2B-AR subtype in MDCKII cells: random delivery to apical and basolateral surfaces with rapid (t(1/2) < or = 60 min) apical versus slower (t(1/2) = 10-12 h) basolateral turnover. Apical delivery of a spinophilin subdomain containing the alpha 2-AR-interacting region (Sp151-483) by fusion with apically targeted p75NTR extended the half-life of alpha 2B-AR at the apical surface to approximately 3.6 h and eliminated the rapid phase (0-60 min) of alpha 2B-AR turnover on that surface. Furthermore, we examined alpha 2B-AR turnover at the surface of mouse embryo fibroblasts derived from wild type (Sp+/+) or spinophilin knock-out (Sp-/-) mice. Two independent experimental approaches demonstrated that agonist-evoked internalization of HA-alpha 2B-AR was accelerated in mouse embryo fibroblasts derived from Sp-/- mice. These findings are consistent with the interpretation that endogenous spinophilin contributes to the stabilization of alpha 2B-AR and presumably all three alpha2-AR subtypes at the surface of target cells and may act as a scaffold that could link alpha 2-ARs to proteins interacting with spinophilin via other domains.
Highlights
Madin-Darby canine kidney (MDCKII) cells at steady state
Redirection of a Spinophilin Subdomain to the Apical Surface of MDCKII Cells—To test the hypothesis that spinophilin contributes to cell surface ␣2-Adrenergic receptors (ARs) retention, we explored whether redirection of the receptor-interacting domain of spinophilin to the apical surface of polarized MDCKII cells would lead to enhanced apical retention of randomly delivered ␣2BAR
The present studies demonstrate a role for spinophilin in the stabilization/retention of the ␣2B-AR at the cell surface both in a cultured cell system and in cells derived from spinophilin knock-out mice
Summary
Transwell culture chambers (0.4-m pore size) were purchased from Costar (Cambridge, MA). NHS-SS-Biotin and Immunopure Immobilized Streptavidin were purchased from Pierce. Both [35S]EasyTagTM Express Protein Labeling mix (1200 Ci/mmol) and [3H]methoxy-inulin (126.5 mCi/g) were from PerkinElmer Life Sciences. Mouse monoclonal anti-c-Myc (clone 9E10) ascites was purchased from Covance Research Products Inc. Both the mouse anti-gp135 and mouse anti-EGFR were gifts from. Alexa Fluor 488-conjugated fluorescent goat anti-mouse, goat anti-rat, and goat anti-rabbit IgG were purchased from Molecular Probes (Eugene, OR). Cy3-conjugated donkey anti-mouse IgG was purchased from Jackson Immunochemicals. Sheep anti-mouse, donkey anti-rabbit, and goat anti-rat horseradish peroxidase-conjugated IgG were purchased from Amersham Biosciences. The rat p75NTR cDNA was a generous gift from Dr Bruce Carter (Department of Biochemistry, Vanderbilt University). The retroviral vector pBabe-HA-␣2B-AR was kindly provided by Drs Dan Gil and John Donello (Allergan, Irvine, CA)
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