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Departments of Pharmacology (A.E.B., Q.W., L.E.L.) and Molecular Physiology and Biophysics (M.R.), Vanderbilt University Medical Center, Nashville, Tennessee
Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York (P.G.)
Department of Psychiatry, Yale University School of Medicine, New Haven, Connecticut (P.B.A.)
Abstract
Agonist activation regulates reciprocal interactions of spinophilin and arrestin with the 2A- and 2B -adrenergic receptor (AR) subtypes via their 3i loop. Because arrestin association with G protein-coupled receptor is preceded by redistribution of arrestin to the cell surface, the present studies explored whether agonist activation of the 2A- and 2B -AR subtypes also led to spinophilin enrichment at the cell surface. Live cell imaging studies using a green fluorescent protein-tagged spinophilin examined spinophilin localization and its regulation by 2 -AR agonist. Agonist activation of 2A-AR preferentially, compared with the 2B-AR, led to spinophilin enrichment at the cell surface in human embryonic kidney 293 cells and in mouse embryo fibroblasts derived from spinophilin null mice. Activation of the LEESSSS2A-AR, which has enriched association with spinophilin compared with the wild-type (WT) 2A-AR, does not show an enhanced redistribution of spinophilin to the surface compared with WT 2A-AR, demonstrating that the ability or affinity of the receptor in binding spinophilin may be independent of the ability of the receptor to effect spinophilin redistribution to the surface. Agonist-evoked enrichment of spinophilin at the cell surface seems to involve downstream signaling events, manifested both by the pertussis toxin sensitivity of the process and by the marked attenuation of spinophilin redistribution in cells expressing the -adrenergic receptor kinase-C tail, which sequesters subunits of G proteins. Together, the data suggest that agonist-evoked spinophilin enrichment at the cell surface is caused by receptor-evoked signaling pathways and is independent of the affinity of the receptor for the spinophilin molecule.
The 2-adrenergic receptors (ARs) are members of the large superfamily of G protein-coupled receptors. There are three 2-AR subtypes (2A, 2B, and 2C), each of which is activated by the endogenous catecholamines epinephrine and norepinephrine and performs multiple physiological functions via pertussis toxin-sensitive Gi/Go proteins (Limbird, 1988). Cellular signaling pathways regulated by the 2A-AR subtype in native cells include inhibition of adenylyl cyclase, activation of receptor-operated K+ channels, inhibition of voltage-gated Ca2+ channels, and activation of the mitogen-activated protein kinase cascade (Limbird, 1988; Kobilka, 1992; Richman and Regan, 1998).
Regions of the 3i loops of the 2A- and 2B-AR subtypes not implicated in G protein coupling have been demonstrated to be critical for stabilization of these subtypes at the basolateral surface of polarized renal epithelial cells in culture (Edwards and Limbird, 1999). A search for proteins localized at or near the cell surface that interact with the 3i loop of the 2-AR and thus could be responsible for this stabilization resulted in the identification of the protein spinophilin (Richman et al., 2001). Spinophilin (Allen et al., 1997; Satoh et al., 1998) is a ubiquitously expressed, multidomain-containing protein that possesses domains for F-actin binding, protein phosphatase 1 (PP1) binding, a single PDZ domain, and three coiled-coil domains. Spinophilin is endogenously enriched under the basolateral domain of cultured renal epithelial cells, Madin-Darby canine kidney (MDCKII) cells (Satoh et al., 1998; Richman et al., 2001). Brady et al. (2003) demonstrated that spinophilin does, in fact, contribute to stabilization of 2-AR at the cell surface. In addition to its GPCR-interacting domain (Smith et al., 1999; Richman et al., 2001; Wang and Limbird, 2002), the other domains of spinophilin may allow for the formation of multiprotein complexes in intact cells that contribute to receptor localization and signaling complex formation.
Recent studies have shown that spinophilin and arrestin share regions of interaction in the 3i loop of 2A-AR and 2B-AR and that agonist occupancy of these receptors enhances spinophilin as well as arrestin association with the receptor (Wang and Limbird, 2002). Because agonist-induced association of arrestin with GPCR is preceded by agonistenhanced translocation of arrestin to the cell surface, and because arrestin versus spinophilin interactions with either the 2A-AR or 2B-AR subtypes are reciprocal in nature (Wang et al., 2004), the present study examines whether 2-AR activation enriches spinophilin localization at the cell surface, using spinophilin-GFP fusion proteins to monitor spinophilin localization in live cells over time.
Materials and Methods
Materials. Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) was prepared by the Cell Culture Core, a facility sponsored by the Diabetes Research and Training Center at Vanderbilt University Medical Center. Fetal calf serum was purchased from Atlanta Biologicals (Norcross, GA). The pEGFP-C1-spinophilin cDNA was a gift from Roger J. Colbran (Department of Molecular Physiology and Biophysics, Vanderbilt University). The retroviral vector pLEGFP-N1 was purchased from BD Biosciences Clontech (Palo Alto, CA). The retroviral vectors pBabe-HA-2A-AR and pBabe-HA-2B-AR were kindly provided by Drs. Dan Gil and John Donello (Allergan, Irvine, CA). Poly-D-lysine, polybrene, and puromycin were purchased from Sigma-Aldrich (St. Louis, MO), and pertussis toxin was from List Biological Laboratories Inc. (Campbell, CA). UK-14,304 was ordered from Sigma/RBI (Natick, MA). MatTek dishes were purchased from MatTek (Ashland, MA). Rat anti-HA 3F10 high affinity was from Roche Diagnostics (Indianapolis, IN). Spinophilin (aa 286eC390) antibody was from Dr. Colbran (Department of Molecular Physiology and Biophysics, Vanderbilt University) and purified by us (Richman et al., 2001).
Cell Culture. HEK 293 cells were maintained in DMEM supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 10 e/ml streptomycin at 37°C, 5% CO2. Mouse embryo fibroblasts (MEFs) were isolated from spinophilin knockout (SpeC/eC) mice (Feng et al., 2000) as described previously (Brady et al., 2003). MEFs were immortalized via standard NIH3T3 protocol (Todaro and Green, 1963). Cells were cultured in DMEM supplemented with 10% fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 10 e/ml streptomycin at 37°C, 5% CO2.
Transfection or Transduction of Cells. HEK 293 cells were transfected using FuGENE 6 reagent (Roche Diagnostics) according to the manufacturer's direction with a pCMV4 vector backbone (control) or pCVM4-HA-2A AR. The pCMV4 vector for this receptor has been described previously (Schramm and Limbird, 1999). The cDNA encoding the C terminus of the G protein receptor kinase 2 (GRK2), known as the -adrenergic receptor kinase (ARK)-C tail, was obtained from Marc Caron (Duke University, Durham, NC).
Immortalized SpeC/eC MEFs were transduced with retroviral vectors encoding HA-tagged 2-AR receptors and GFP-tagged spinophilin constructs as described previously (Brady et al., 2003). Transduced cells were selected for pBabe-2-AR expression by treatment for 36 h with 4 e/ml puromycin (the pBabe retroviral vector carries the resistance gene for puromycin). Stable 2-AR-expressing clones were isolated by standard ring-cloning methods and screened via radioligand binding analysis, using the radiolabeled 2-AR-antagonist [3H]rauwolscine, essentially as described previously (Edwards and Limbird, 1999). To assess the localization of spinophilin in live cells, either holo-spinophilin (Sp1-817) or spinophilin amino acids 151 to 444 were amplified via polymerase chain reaction extension from wild-type spinophilin using primers engineered to contain unique restriction sites HindIII and SalI. The amplified polymerase chain reaction product was then subcloned into the pLEGFP-N1 retroviral backbone at the HindIII and SalI sites. The final constructs were verified by sequencing analysis. MEFs stably expressing HA-2A- or HA-2B-AR were then transduced with the pLEGFP-spinophilin retroviral constructs and selected by growth in 500 e/ml G418 (Geneticin).
Determination of Receptor Density. Permanent transformants of MEFs expressing either the HA-2A- or HA-2B-AR were assayed for functional receptor density using standard saturation binding protocols using [3H]rauwolscine (HA-2A-AR) (PerkinElmer Life and Analytical Sciences, Boston, MA) or [3H]RX 821002 radioligand (HA-2B-AR) (PerkinElmer Life and Analytical Sciences) (Edwards and Limbird, 1999) and nonlinear regression analysis using Prism (GraphPad Software Inc., San Diego, CA). The density of the MEFs expressing the HA-2A-AR was 2.0 pmol/mg membrane protein, and the density of the MEFs expressing the HA-2B-AR was 1.4 pmol/mg membrane protein.
Live Cell Imaging. HEK 293 or MEFs were plated the night before the assay on MatTek dishes (35 mm) coated with 2.5 e/cm2 poly-D-lysine at 3.5 x 105 cells per dish in medium containing the 2-AR antagonist phentolamine (10eC6 M) to eliminate effects of catecholamines that might be present in the serum-containing DMEM. The day of the assay, cells were washed 2 x 30 min in serum-free DMEM containing 0.01% bovine serum albumin and 3 x 30 min in serum-free DMEM supplemented with 20 mM HEPES. All washes were performed at 37°C. Quantitative, live cell confocal microscopy was performed using a Zeiss LSM510 confocal microscope equipped with a 488-nm argon/krypton laser and fitted with a heated and humidified chamber system. All experiments were performed at 37°C using a 40x/1.3 numerical aperture oil immersion lens. Emitted fluorescence was detected with a 550-nm-long pass filter.
Data capture was carried out in the following manner. First, a z-stack of the cell of interest was acquired before drug treatment, and then the time series was begun, where sequential images at a single, central plane were captured at 1-min intervals for the duration of the time course. The indicated ligand was introduced immediately after the first scan. After the completion of the time course, usually 10 min in the presence of UK-14,304, a post-drug z-stack was acquired (specific incubation conditions are given in the figure legends). GFP-spinophilin redistribution after drug treatment was reflected by an increase in plasma membrane fluorescence, quantified as changes in pixel intensity. Using MetaMorph software (Universal Imaging Corporation, Downingtown, PA), measurements were made by selecting a region encompassing the entire plasma membrane (defined as total), and then selecting a region just inside the plasma membrane (defined as inside). The difference between these two measurements reflects the defined membrane area. The product of the number of pixels and the average pixel intensity was calculated for the total area as well as for the defined membrane area for each cell both before (t = 0) and after (t = 10 min) drug treatment. The membrane pixel intensity was then expressed as a fraction of total pixel intensity. Changes in fluorescence over time are expressed as percentage of time 0.
Coimmunoisolation of HA-2AR with Spinophilin. Coimmunoisolation of HA-2AR subtypes with spinophilin was performed as described previously (Wang and Limbird, 2002) with a few modifications. MEFs transduced with HA-2AR subtypes were stimulated with or without agonist (100 e epinephrine + 1 e propranolol) for 5 min. Endogenous spinophilin was detected with spinophilin antibodies in immunocomplexes isolated by rat anti-HA antibodies.
Results
Enrichment of GFP-Spinophilin at the Plasma Membrane of HEK 293 Cells Mediated by 2A-AR. Previous findings from our laboratory have demonstrated that all three 2-AR subtypes (2A, 2B, and 2C) interact with the multidomain protein spinophilin via their third intracellular loop (Richman et al., 2001). Interactions of the 2A-AR and 2B-AR subtypes with spinophilin are enhanced by agonist, as revealed by the enrichment of spinophilin in 2-AR-containing immunoisolates after treatment of target cells with an 2-AR agonist (Richman et al., 2001; Wang and Limbird, 2002). This led us to the hypothesis that the increase in 2-AR-associated spinophilin detected in coimmunoisolation assays might be due, at least in part, to an agonist-induced enrichment of spinophilin at the plasma membrane, by analogy with agonist-induced redistribution of another 3i loop-interacting protein to the cell surface, arrestin (Barak et al., 1997; Groarke et al., 1999). Although we did observe some increase in the membrane localization of endogenous spinophilin in both MEFs and SCG neurons (data not shown), the apparent magnitude of the change was not equivalent in every cell, and by using antibody staining, we could not analyze the same cell before and after drug treatment, because we had to fix and permeabilize the cell preparations to identify endogenous spinophilin. Thus, to evaluate agonist-evoked spinophilin redistribution in a more quantitative manner within single cells (using each cell as its own control), a cDNA encoding a GFP-spinophilin fusion protein was coexpressed with 2A-AR in HEK 293 cells, and the localization of GFP-spinophilin was monitored in real-time by confocal microscopy after stimulation of the HEK 293 cells with the 2-AR agonist UK-14,304. Figure 1A provides a schematic diagram of N-terminal fusion of GFP with the multidomain protein spinophilin.
Unlike for arrestin, which is principally cytosolic (Barak et al., 1997), a population of spinophilin seems to constitutively associate at or just underneath the surface membrane as well as in a cytosolic pool (Satoh et al., 1998; Richman et al., 2001). Our data indicate that, under basal conditions, 28.2 ± 1.5 and 28.3 ± 2.6% of the total cellular GFP-spinophilin is localized to the cell membrane in HEK 293 cells transfected with pCMV4 alone or pCMV4 encoding the 2A-AR, respectively. The preexistence of a considerable fraction of spinophilin at the cell surface masks the quantitative extent of spinophilin redistribution using visual inspection alone (Fig. 1B), but quantitative confocal microscopy analysis (see Materials and Methods) reveals a 30% increase in GFP-spinophilin at the cell surface after agonist activation of the 2A-AR (Fig. 1C). No detectable increase is observed in cells expressing the control vector pCMV4. This redistribution of GFP-spinophilin to the plasma membrane was detected as early as 2 min after the addition of agonist and did not seem to increase further after 10 min; nor did we observe a reversal of the association with longer time points (i.e., 30 min; data not shown). It is interesting that activation of the LEESSSS2A-AR, which has enriched association with spinophilin compared with the WT 2A-AR when examined in immunoisolation experiments (Wang and Limbird, 2002), does not show an enhanced redistribution of spinophilin to the surface compared with WT 2A-AR (Fig. 1C), and—if anything—is slightly attenuated in its effectiveness in effecting spinophilin redistribution. These data suggest the possibility that receptor-spinophilin complex formation may be an event independent of agonist-induced spinophilin enrichment at the cell surface.
It was of interest to us in our preliminary studies that a similar redistribution of spinophilin was not seen after agonist activation of the 2B-AR expressed in HEK 293 cells. However, because we were not able to achieve similar expression of the 2B-AR subtype as the 2A-AR subtype after transient transfection in these cells, we were concerned that receptor density, per se, might be responsible for this apparent subtype-selective response, and we chose to pursue possible subtype selectivity of this response in permanent, clonal cell lines.
Agonist-Mediated Redistribution of Spinophilin-GFP in SpeC/eC MEFs. We chose to explore the possible subtype selectivity of 2-AR-mediated enrichment of spinophilin at the cell surface in MEFs derived from mice null for spinophilin (SpeC/eC) expressing either the 2A-AR (2.0 pmol/mg protein) or the 2B-AR (1.4 pmol/mg protein) in clonal cell lines selected after retroviral transduction with either receptor subtype. Selection of the SpeC/eC genetic background to explore real-time distribution of spinophilin-GFP was intended to increase the sensitivity of our signal.
Agonist activation of the 2A-AR subtype in MEFs evokes a statistically significant increase in cell surface-associated spinophilin-GFP (Fig. 2B). Statistically significant increase in cell surface-associated spinophilin-GFP, however, is not observed after agonist activation of the 2B-AR subtype in this same cellular background (Fig. 2C), even though agonist activation increases the association of 2B-AR with spinophilin as detected in coimmunoisolation assays (Fig. 2D), a finding that shows that agonist occupancy of the 2B-AR can elicit its characteristic association with spinophilin. These data suggest that, even in cells expressing the 2B-AR at densities comparable with those of the 2A-AR, the redistribution of spinophilin is more readily detectable in cells expressing the 2A-AR subtype and that redistribution of spinophilin to the surface may be independent of the affinity of the receptor subtype for spinophilin, which is further revealed by studies with solely the receptor binding domain of spinophilin (see below).
For the studies in MEFs, a C-terminal GFP fusion with spinophilin was used (Fig. 2A), simply because the construction of the retroviral vector for transduction of these cells was more easily achieved in this configuration (Fig. 2A). The comparable findings using either N- or C-terminal GFP-spinophilin fusion proteins (cf. Fig. 1C with Fig. 2B) affirm that the present observations are not attributable to steric properties defined by the locus of the GFP fusion.
The Receptor Binding Domain of Spinophilin Is Not Sufficient for Agonist-Induced Enrichment at the Plasma Membrane. As a means to ascertain whether the receptor binding domain of spinophilin is sufficient for agonist-induced redistribution of spinophilin to the cell surface, or whether other domains are necessary, we expressed just the receptor binding domain of spinophilin (aa 151eC444) (Fig. 3A) as a GFP fusion protein. In contrast to agonist-evoked enrichment of holo-spinophilin-GFP at the cell surface, no such enrichment occurred after UK-14,304 treatment of cells expressing Sp151-444-GFP (Fig. 3B). These findings suggest that receptor-spinophilin protein-protein interactions per se are not sufficient to permit the agonist-stimulated enrichment of spinophilin at the cell surface, implying that other domains of spinophilin, and thus other mechanisms, are involved, including the participation of downstream signaling pathways.
Agonist-Mediated Redistribution of GFP-Spinophilin to the Plasma Membrane Requires G. We were curious whether 2A-AR interaction with its cognate G protein Gi/Go was critical for enrichment of spinophilin at the cell surface after agonist activation. To test this hypothesis, MEFs were incubated with 100 ng/ml pertussis toxin overnight to ADP-ribosylate MEF Gi and thus disrupt the ability of the 2A-AR to interact with the G protein. As can be seen in Fig. 4A, pertussis toxin treatment diminished the extent of 2A-AR-mediated spinophilin redistribution to the cell surface after UK-14,304 treatment of either HEK 293 cells or MEFs. Parallel studies demonstrate that the G protein-dependent mitogen-activated protein kinase signaling activated by the 2A-AR in HEKs and MEFs also is attenuated by pertussis toxin treatment, confirming that toxin exposure of the cells was successful in modifying at least a fraction of the Gi- and perturbing Gi-dependent functions. These findings suggest either that receptor-G protein interactions per se or receptor-evoked signaling pathways contribute to the enrichment of spinophilin at the cell surface after agonist activation of the receptor.
To further address the possible role of G proteins in 2A-AR-evoked spinophilin enrichment at the cell surface, we overexpressed the C-terminal domain of ARK1 (or GRK2) to sequester the subunits of G proteins (Koch et al., 1994). We chose this approach because we have recently observed that spinophilin association with the 2A-AR seems to recognize a complex containing the receptor and the subunits of heterotrimeric G proteins, by analogy with the basis for GRK association with the agonist-activated GPCRs (Wang et al., 2004). The findings in Fig. 4B suggest that spinophilin enrichment at the cell surface after agonist treatment of cells also involves G subunits, because expression of the -interacting domain of -adrenergic receptor kinase, its C terminus (ARK-C tail), disrupts the ability to detect agonist-evoked spinophilin enrichment at the cell surface in HEK 293 cells. These data, then, emphasize the importance of G protein activation in 2A-AR-mediated enrichment of spinophilin at the cell surface.
Discussion
The present study demonstrates three important findings. First, agonist activation of the 2A-AR enriches spinophilin association with the plasma membrane, a finding that has not been reported previously for GPCRs that interact with spinophilin. Second, redistribution to the cell surface involves the subunits of the pertussis toxin-sensitive G protein Gi. This finding means either that receptor-elicited signaling via -mediated pathways is involved in spinophilin enrichment at the cell surface or that a receptor- complex, enriched by agonist activation of the receptor, serves as the "docking site" for spinophilin, or both. Third, the ability of 2-AR subtypes to associate with spinophilin is independent of agonist-evoked spinophilin enrichment at the cell surface. Several lines of evidence are consistent with this interpretation. First, the observation that the receptor-interacting domain of spinophilin, Sp151-444, is insufficient to respond to agonist activation of the 2A-AR for redistribution to the surface (Fig. 3) is consistent with 2A-AR downstream signaling events contributing to agonist-enrichment of spinophilin at the plasma membrane, perhaps events involving phosphorylation and/or dephosphorylation of the other domains of spinophilin, including the actin binding, PP1 regulatory, PDZ, or coiled-coil domains. Second, stimulation of an 2A-AR mutant lacking the GRK2 phosphorylation consensus sequence does not result in a greater enrichment of spinophilin at the cell surface than for WT 2A-AR (Fig. 1C), despite the fact that this mutated receptor displayed an increased association with spinophilin in coimmunoisolation studies compared with WT 2A-AR (Wang and Limbird 2002). Third, activation of the 2B-AR does not lead to detectable redistribution of spinophilin (Fig. 2C), in contrast to findings for 2A-AR, although the 2B-AR readily complexes with spinophilin in MEFs (cf. Figure 2D) and in other cells. It is possible, of course, that both the 2A-AR and 2B-AR can effect spinophilin redistribution to the membrane, but the sensitivity of our measurements preclude detection of statistically significant redistribution in response to agonist activation of the 2B-AR. Nonetheless, these findings indicate that there is a preferential capability of the 2A-AR to effect redistribution of spinophilin to the cell surface. Because both 2A-AR and 2B-AR seem to couple to the similar G proteins and effector molecules, the apparently differential capacities of these subtypes to recruit spinophilin to the plasma membrane may be a mechanism contributing to subtype signaling diversity. For example, in dendritic spines of neurons, where spinophilin is enriched (Allen et al., 1997), the 2A-AR subtype may be able to engage or amplify signaling pathways using spinophilin-associated proteins in a way that the 2B-AR cannot.
Future studies will reveal the generality of this finding for other GPCRs, especially for those that have already been shown to interact with spinophilin (Smith et al., 1999), and their functional relevance in vitro and in vivo.
Acknowledgements
We thank Carol Ann Bonner and Sean B. Schaffer for excellent and dedicated technical support, and Drs. David Piston and Sam Wells (Department of Molecular Physiology and Biophysics, Vanderbilt University) for considerable technical advice.
doi:10.1124/mol.104.005215.
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