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«Downloaded from by guest on May 7, 2016 Limbird Departments of Pharmacology and of *Molecular Physiology and Biophysics, Center ...»

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Spinophilin Stabilizes Cell Surface Expression of α2B-Adrenergic Receptors

Ashley E. Brady, Qin Wang, Roger J. Colbran*, Patrick B. Allen§, Paul Greengard†, Lee E.

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Departments of Pharmacology and of *Molecular Physiology and Biophysics, Center for

Molecular Neuroscience, Vanderbilt University Medical Center, Nashville, TN 37232-6600,


Department of Psychiatry, Yale University School of Medicine, New Haven, CT 06508, and † Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, NY Running Title: Role for Spinophilin in α2B-AR Cell Surface Retention Corresponding author: Lee E. Limbird Department of Pharmacology Vanderbilt University Medical Center 464 Robinson Research Building Nashville, TN 37232-6600 (615) 343-3538 FAX: (615) 343-7286 E-mail: lee.limbird@vanderbilt.edu


The third intracellular (3i) loops of the α2A-(Keefer, J. R., et al. (1994) J.Biol.Chem. 269, 16425) and α2B-(Saunders, C. and Limbird L.E. (2001) Mol.Pharmacol. 57, 44) adrenergic receptor subtypes are critical for retention of these receptors at the basolateral surface of polarized MadinDarby canine kidney (MDCKII) cells at steady state. The 3i loops of the α2A, α2B, and α2C-AR subtypes interact with spinophilin, a multidomain protein that, like the three α2-AR subtypes, is enriched at the basolateral surface of MDCKII cells (Richman, J. G., et al. (2001) J.Biol.Chem.

276, 15003; Nakanishi, H., et al. (1997) J.Cell Biol. 139, 951). The present studies provide Downloaded from http://www.jbc.org/ by guest on May 7, 2016 evidence that α2-AR interaction with spinophilin contributes to cell surface stabilization of the

receptor. We exploited the unique targeting profile of the α2B-AR subtype in MDCKII cells:

random delivery to apical and basolateral surfaces with rapid (t1/2 ≤ 60 min) apical versus slower (t1/2 = 10-12 hr) basolateral turnover (Wozniak, M. and Limbird, L. E. (1996) J.Biol.Chem. 271, 5017). Apical delivery of a spinophilin subdomain containing the α2-AR-interacting region (Sp151-483) by fusion with apically targeted p75NTR extended the half-life of α2B-AR at the apical surface to ≅3.6 hr and eliminated the rapid phase (0-60 min) of α2B-AR turnover on that surface. Furthermore, we examined α2B-AR turnover at the surface of mouse embryo fibroblasts (MEFs) derived from wildtype (Sp+/+) or spinophilin knock-out (Sp-/-) mice. Two independent experimental approaches demonstrated that agonist-evoked internalization of HA-α2B-AR was accelerated in MEFs derived from Sp-/- mice. These findings are consistent with the interpretation that endogenous spinophilin contributes to the stabilization of α2B-AR, and presumably all three α2-AR subtypes, at the surface of target cells and may act as a scaffold that could link α2-ARs to proteins interacting with spinophilin via other domains.


α2-adrenergic receptors (α2-ARs) are members of the large superfamily of G-protein coupled receptors that contain seven putative transmembrane spanning regions. There are three α2-AR subtypes (α2A, α2B, and α2C), each of which is activated by the endogenous catecholamines, epinephrine and norepinephrine, and performs multiple physiological functions via pertussis toxin sensitive Gi/Go proteins (1). Cellular signaling pathways regulated by α2A-AR in native cells include inhibition of adenylyl cyclase, activation of receptor–operated K+

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protein kinase (MAPK) cascade (1-3). Many cells that express α2-ARs are polarized, including renal and intestinal epithelia, where the α2-AR serves to regulate sodium and water resorption (4,5), as well as neurons, where these receptors act to suppress neurotransmitter release (6). The physiological functions mediated by α2-ARs in polarized cells are dependent upon precise localization of the receptor at the basolateral surface in order to gain access to neural- and blooddelivered catecholamines.

The α2-AR subtypes demonstrate unique targeting and retention profiles in polarized renal epithelial Madin-Darby canine kidney (MDCKII) cells in culture (7). Previous work in our laboratory has shown that the α2A-AR subtype is directly targeted to the basolateral surface, where it exhibits a half-life of 10-12 hr (8). Direct and exclusive basolateral targeting of α2A-AR was found to be dependent upon several noncontiguous regions within or near the bilayer, while retention of the receptor at the basolateral surface appears dependent upon the third intracellular (3i) loop (8). Deletion of the 3i loop results in accelerated surface turnover (t1/2 ≅ 4.5 hr) of the α2A-AR at the basolateral surface (9). Unlike the α2A-AR, the α2B-AR subtype is randomly targeted to both the apical and basolateral subdomains, and then selectively retained at the basolateral surface of polarized MDCKII cells, where the receptor has a half-life comparable to that of the α2A-AR subtype (t1/2 ≅10-12 hr) (7). Like for the α2A-AR, the 3i loop of the α2B-AR also is critical for basolateral surface stabilization of this subtype (10). In contrast to stable retention of the α2B-AR on the basolateral surface, the half-life on the apical surface is estimated to be dramatically shorter, on the order of minutes (7). Taken together, these data suggest that the stabilization/retention of α2A- and α2B-AR at specific membrane domains is most likely mediated through interactions of the 3i loop with other proteins either within or underlying the

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proteins and has led to the identification of two α2-AR-interacting molecules: 14-3-3ζ (11), and spinophilin (12). Spinophilin is an 817 amino acid, ubiquitously expressed, multi-domaincontaining protein with an apparent Mr on SDS-PAGE of approximately 130kDa. It was originally identified both as a protein phosphatase 1 (PP1)-binding protein localized to dendritic spines, hence the name spinophilin (13), as well as an F-actin-binding protein (14). Spinophilin (also known as neurabin II), is highly homologous to the brain-specific protein, neurabin I (14).

In addition to the domains described above, spinophilin contains a single PDZ domain and three coiled-coil domains at the C-terminus, the latter of which mediate homo-multimerization in vitro (14) and may allow for the formation of multi-protein complexes in intact cells. Spinophilin previously was identified as a D2 dopamine receptor (D2R)-interacting protein using the 3i loop of the D2R as bait in a yeast two-hybrid screen (15). The D2R-binding domain in spinophilin (residues 151-444), located between the F-actin binding domain and the PP1 regulatory domain, also interacts with all three of the α2-AR subtypes (12), and will be referred to as the receptorinteracting domain. Because reports in the literature, as well as our own observations, indicate that spinophilin is specifically enriched at the basolateral surface of polarized epithelial cells (12,15,16), we postulate that spinophilin may be involved in tethering and/or stabilizing the receptor at the cell surface via interactions with the α2-AR 3i loops.

The present studies utilized two different biological systems to explore the role of spinophilin in α2-AR stabilization at the cell surface. First, the unique targeting profile of the α2B-AR subtype in polarized MDCKII cells (random delivery with rapid turnover at the apical

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spinophilin to the apical surface of polarized MDCKII cells would result in enhanced apical retention of randomly delivered α2B-AR. Second, the role of spinophilin in α2-AR surface turnover was addressed by studying the internalization profile of the α2B-AR in mouse embryonic fibroblasts (MEFs) derived from wild type (Sp+/+) or spinophilin knock-out (Sp-/-) mice. The findings from both lines of investigation implicate spinophilin in the stabilization of the α2B-AR at the cell surface.


Materials Transwell culture chambers, 0.4 µm pore size, were purchased from Costar, Cambridge, MA. Doxycycline hydrochloride was purchased from Sigma chemicals. NHS-SS-Biotin and Immunopure Immobilized Streptavidin were purchased from Pierce. Both [35S] EasyTag™ Express Protein Labeling mix (1200 Ci/mmol) and [3H]methoxy-inulin (126.5 mCi/g ) were from NEN Life Science Products, Inc. Cysteine and methionine-free DMEM was from Cellgro Mediatech. Dulbecco’s modified Eagle’s medium was prepared by the Cell Culture Core, a

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Medical Center. Fetal calf serum was purchased from Sigma. Mouse monoclonal 12CA5 antibody against the HA epitope was obtained from BABCo. Affinity matrix-coupled high affinity rat monoclonal anti-HA antibody (clone 3F10), rat monoclonal anti-HA (clone 3F10) and mouse monoclonal HA.11 (clone 16B12) antibodies were purchased from Roche. Mouse monoclonal anti-c myc (clone 9E10) ascites was purchased from Covance Research Products Inc., Denver, PA. Both the mouse anti-gp135 and mouse anti-EGFR were gifts from Peter J.

Dempsey (Department of Pathology, University of Washington, Harborview Medical Center, Seattle, Washington). Rabbit anti-spinophilin antibody, raised against spinophilin amino acids 286-390 was purified in our lab (for details see (12,17)). 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 (HRP) conjugated IgG were purchased from Amersham Biosciences. The rat p75NTR cDNA was a generous gift from Dr. Bruce Carter (Dept. of Biochemistry, Vanderbilt University). The retroviral vector, pBabe-HA-α2B-AR was kindly provided by Drs. Dan Gil and John Donello (Allergan, Irvine, CA).

MDCKII Cell Culture and Polarization MDCKII cells were plated at a density of 1.2 X106 cells per 100 mm polycarbonate membrane filter (Transwell culture chambers, 0.4 µm pore size), and cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (Sigma) and 100 U/mL penicillin and 10 µg/mL streptomycin at 37ºC/5% CO2 with medium changes every other

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functionally polarized phenotype, as described previously (18). Leak assays of [3H]methoxyinulin were performed as described previously (19) prior to each half-life experiment to verify that the MDCKII cells had developed tight junctions and that the apical and basolateral compartments were functionally separated.

Generation of cDNAs Encoding Myc-p75-spinophilin Fusion Proteins The cDNA encoding the pTRE-myc-p75-Sp151-483 fusion protein was generated via overlapping PCR extension using PFU turbo DNA polymerase (Stratagene). The myc tag was inserted 5’ to the coding start site of full length rat p75NTR (14) and 3’ of the N-terminal cleavable signal sequence. Four glycine residues (gly4) were engineered via PCR onto the Cterminus of p75NTR with the intention of permitting independent folding of the spinophilin subdomain and decreased steric hindrance for interacting with other potential binding partners.

The pTRE cDNA backbone (Clontech) has a tetracycline inducible promoter that is intended to confer regulated expression of the fusion construct by treatment with the synthetic tetracycline analog, doxycycline. Two fusion proteins were generated. Myc-p75-Sp151-483 includes the receptor binding domain and the PP1 regulatory domain of the full-length spinophilin, while myc-p75-Sp151-586 also contains the PDZ binding domain. (cf. schematic of spinophilin domain structure in Fig. 2B) The cDNAs were sequenced in their entirety via 33P-Thermo Sequenase Radiolabeled Terminator Cycle Sequencing Kit (USB Corporation, Cleveland, OH) to confirm that the sequences were correct.

Creation of Clonal MDCKII Cell Lines Expressing the Myc-p75-spinophilin Fusion Protein Permanent clonal cell lines were developed in MDCKII cells using FuGENE-6

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linearized pTET-On plasmid (Clontech) was co-transfected with 6 µg of pTRE-myc-p75Spinophilin construct and 1 µg of a vector encoding a hygromycin resistance gene into MDCKII cells already stably expressing the HA-α2B-AR, generated as described previously (7). Stably transfected cells were selected through growth in 400µg/mL hygromycin and assayed for HAα2B-AR expression via radioligand binding analysis using the antagonist [3H]-rauwolscine (20) and for myc-p75-spinophilin expression and inducibility via Western analysis and immunofluorescence using anti-c-myc antibody. Despite the use of an inducible expression system, myc-p75-spinophilin expression occurred even in the absence of doxycycline.

Nonetheless, cells were treated overnight with 1 µg/mL doxycycline before the day of the experiment to assure maximal myc-p75-spinophilin expression.

Immunofluorescence in Polarized MDCKII Cells Polarized MDCKII cells stably expressing HA-α2B-AR were grown on 12 mm Transwells for 5-7 days (as described above) and prossessed as described previously for immunolocalization of HA-α2B-AR and endogenous spinophilin (12) and for detection of endogenous apical (gp135) and basolateral (EGF Receptor) marker proteins (21) (Fig. 1).

MDCKII cells expressing myc-p75-Sp151-483 fusion protein in the HA-α2B-AR background were treated with 1µg/mL doxycycline overnight to maximize expression of myc-p75-Sp151prior to staining. All steps were performed essentially as described previously (21) except

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the detection of HA-α2B-AR (Fig. 2D).

Immunoprecipitation of Apically Targeted Myc-p75-spinophilin Fusion Proteins from Stable MDCKII Cell Lines MDCKII cells lines stably expressing the HA-α2B-AR alone or myc-p75-Sp151or myc-p75-Sp151-586 in the HA-α2B-AR background were grown to confluence in 100 mm tissue culture dishes. All dishes were treated with 1 µg/mL doxycycline for 16 hr before harvesting the cells to maximize fusion protein expression. On the day of the assay, dishes were washed twice with Dulbecco’s phosphate buffered saline supplemented with 1mM MgCl2 and

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