INTRODUCTION

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VARIOUS RECEPTORS, including -adrenergic receptors (-AR), regulate important functions in adipocytes through the generation of cAMP and the activation of protein kinase A (PKA) (9), and growing evidence suggests that the PKA signaling pathway is functionally compartmentalized in adipocytes. For example, catecholamine activation of lipolysis in both brown and white adipocytes requires far lower levels of cAMP than does activation by forskolin, suggesting that adrenergic receptors more effectively target cAMP generation to activation of lipolysis (12). The molecular basis for the functional compartmentalization of cAMP-mediated responses in adipocytes, however, is presently unknown.

One potential means of physically compartmentalizing cAMP signaling is through the subcellular targeting of PKA. In this regard, A-kinase anchoring proteins (AKAPs) have been proposed to target the holoenzyme to subcellular targets via specific interactions with the regulatory subunit RII (7). RII is most abundantly expressed in brown and white adipose tissue, and targeted disruption of RII produces lean mice that are resistant to obesity (8). Little is known about the subcellular distribution of RII in fat cells or whether interactions with AKAPs tether PKA to specific subcellular locations. The present study was undertaken to determine whether adipose tissue expresses potential AKAPs and whether these proteins are involved in tethering PKA to specific subcellular locations. The C3H/10T1/2 cell line was chosen for this study because these cells can be easily differentiated into adipocytes that exhibit appropriate PKA-mediated responses, including lipolysis to ₃-AR agonists (19). The results show that adipocytes express several proteins that bind RII in vitro. The predominant protein that binds to RII in gel overlay assays was identified as D-AKAP1/S-AKAP84, a putative AKAP that is targeted to mitochondria (6, 11, 14, 15, 18). However, although D-AKAP1 interacts with RII in vitro, the in vivo distribution of these proteins is distinct. Thus D-AKAP1/S-AKAP84 is unlikely to play a role in PKA signaling in adipocytes.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Materials. Paraformaldehyde solution was from Electron Microscopy Sciences (Ft. Washington, PA). Monoclonal antibodies to RII and the catalytic subunit (C) of PKA were from Transduction Laboratories (Lexington, KY). Monoclonal antibody to prohibitin was from RDI (Flanders, NJ). Dr. Susan Taylor (University of California, San Diego) provided polyclonal antibodies to D-AKAP1 and D-AKAP1 core peptide (amino acids 284-408). MitoTracker red and Oregon green-conjugated secondary antibodies were obtained from Molecular Probes (Eugene, OR). All other secondary antibodies were from Jackson Labs (West Grove, PA). Nitrocellulose and 4-15% precast gels were obtained from Novex/Invitrogen (Carlsbad, CA). Tissue culture reagents were from GIBCO (Gaithersburg, MD). All other reagents were from Sigma (St. Louis, MO).

Western blot analysis. Proteins (10-25 µg) were subjected to electrophoresis in a 4-20% precast gel and transferred to nitrocellulose. The nitrocellulose membranes were blocked for 1 h in Tris-buffered saline (TBS) containing 5% milk (Carnation) and 0.1% Tween 20. The blots were incubated with antibodies to D-AKAP1/S-AKAP84 (1:4,000), prohibitin (1:200), RII (1:5,000), or C (1:1,000). After extensive washing in TBS containing 0.1% Tween 20, the blots were incubated in horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody for 1 h. Immunoreactive proteins were visualized by enhanced chemiluminescence (Pierce, Rockford, IL).

Immunocytochemistry. C3H/10T1/2 adipocytes were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4°C for 20 min. Before fixation, some cells were incubated at 37°C for 30 min with 0.1 µM MitoTracker red, a rosamine dye that is selectively incorporated into mitochondria. Fixed cells were quenched in 100 nM glycine, permeabilized in 0.2% Triton X-100, and blocked for 1-2 h in PBS containing 1% each of ovalbumin, bovine serum albumin (BSA), and goat serum. Cells were then incubated either for 1 h with anti-RII antibody or overnight at 4°C with anti-D-AKAP1/ S-AKAP84 antibody. When RII and D-AKAP1/S-AKAP84 immunoreactivity was examined for colocalization, cells were incubated overnight with the two antibodies. The next day, cells were washed three times in PBS and incubated for 1 h in Oregon green- or Cy3-conjugated anti-rabbit or anti-mouse secondary antibody. Control samples were incubated either with secondary antibody alone or with purified RII or the peptide representing the core sequence of D-AKAP1/ S-AKAP84, which was used to generate the D-AKAP1/ S-AKAP84 antibody (14). Fluorescence signals were detected with an Olympus Fluoview laser scanning confocal microscope.

Nuclease protection assay. A fragment of D-AKAP1/ S-AKAP84 cDNA representing the core sequence of D-AKAP1/S-AKAP84 was amplified by PCR and cloned into PCR 2.1 vector, and T7 polymerase was used to generate a probe for nuclease protection assay (NPA). NPA was performed as previously described (5).

Cell culture. C3H/10T1/2 cells were grown in basal Eagle's medium (BME) supplemented with 10% fetal calf serum in 5% CO₂ in air. The medium was changed every 2-3 days. On reaching confluence, cells were placed in differentiating medium that consisted of BME supplemented with 10% fetal calf serum, 1 µM insulin, 1 µM 9-cis-retinoic acid, and 1 µM BRL-49653. Cells were used 8-12 days later, at which time they were over 80% differentiated.

Preparation of total lysates and cell fractionation. Brown and white adipose tissue was obtained from male 200- to 250-g Sprague-Dawley rats (Hilltop, Scottsdale, PA). Total cell lysates were prepared by homogenizing C3H/10T1/2 cells or adipose tissue in TES buffer (50 mM Tris, 2 mM EDTA, and 254 mM sucrose, pH 7.5) containing protease inhibitors (Roche Diagnostics, Mannheim, Germany).

Isolation of mitochondria. Mitochondria were isolated from C3H/10T1/2 adipocytes as described previously (13) with some modifications. Briefly, C3H/10T1/2 adipocytes were homogenized in buffer A (250 mM mannitol, 0.5 mM EGTA, and 5 mM HEPES, pH 7.4) and centrifuged at 3,000 g. Mitochondria were then pelleted by centrifuging the supernatant for 10 min at 10,000 g. The resulting pellet was suspended in a small volume of buffer A, layered on top of 20 ml of 30% (vol/vol) Percoll in 225 mM mannitol, 1 mM EGTA, and 25 mM HEPES (pH 7.4), and centrifuged for 30 min at 95,000 g in a Beckman 60Ti rotor. Mitochondria were collected from the lower part of the dense, brownish yellow mitochondrial band by centrifuging this fraction at 6,300 g.

Expression, purification, and ³²P phosphorylation of RII. The expression plasmid pET11c containing the rat RII (kindly provided by John Scott, Vollum Institute, Oregon Health Sciences University) was transformed into Escherichia coli BL21(DE3) cells (Novagen). RII protein was isolated as described previously (10) with the modification that cells were lysed in buffer containing 0.1% Triton X-100. RII was ³²P phosphorylated by PKA (Calbiochem, La Jolla, CA) as described previously (3, 16).

[³²P]RII overlay assays. Overlay assays were performed as described previously (3, 10, 16). Briefly, proteins in cell lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were then blocked overnight in TBS containing 1% BSA, 5% milk, and 0.05% Tween 20. Blots were then probed with 100,000 cpm/ml of [³²P]RII in blotto for 3-4 h, washed extensively with TBS containing 0.1% Tween 20, and visualized by autoradiography.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

AKAPs are structurally diverse proteins that have been defined by their ability to bind RII in vitro (7). To identify potential AKAPs in adipose tissue, recombinant RII was phosphorylated with [³²P]ATP and used to screen adipocyte extracts in gel overlay assays. RII bound several proteins in brown adipose tissue homogenates, including a prominent 132-kDa band (Fig. 1). The binding of RII to several of these proteins, including the 132-kDa band, was blocked by Ht 31 peptide, a peptide thought to mimic the amphipathic helix of AKAPs and thus serve as a competitive inhibitor of RII-AKAP interaction (7). Binding was not blocked by Ht 31-P peptide, in which the mutation of two proline residues disrupts amphipathic helix formation (Fig. 1). RII also bound to the 132-kDa band in white adipose tissue and in C3H/10T1/2 cells (Fig. 2). In C3H/10T1/2 cells, the 132-kDa protein was strongly induced upon differentiation into adipocytes. In white adipose tissue, RII also bound another lower molecular weight protein that appeared to be the major RII binding protein in this tissue. Although Ht 31 peptide completely blocked the binding of RII to the 132-kDa band, it failed to completely block the binding of RII to this lower molecular weight protein. This protein also was present in C3H/10T1/2 cells, although to a much lesser extent than the 132-kDa band.

View larger version (43K): [in this window] [in a new window]   Fig. 1.   RII overlay analysis of total cellular proteins from brown adipose tissue. Total cell lysates were prepared, subjected to SDS-PAGE, transferred to nitrocellulose, and probed for A-kinase anchoring proteins (AKAPs) as described in METHODS. Blotted proteins were probed with [32P]RII in the presence (+) or absence () of Ht 31 peptide or in the presence of Ht 31-P.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   RII overlay analysis of total cellular proteins from white adipose tissue (A) and C3H/10T1/2 cells (undifferentiated and differentiated adipocytes) (B). Total cell lysates were prepared, subjected to SDS-PAGE, transferred to nitrocellulose, and probed for AKAPs as described in METHODS. Blotted proteins were probed with [32P]RII in the presence or absence of Ht 31 peptide.

The size of the 132-kDa band was similar to that described for D-AKAP1/S-AKAP84 (6, 11, 15, 18). The presence of D-AKAP1/S-AKAP84 in C3H/10T1/2 adipocytes was examined by both immunoblotting and NPA (Fig. 3). An antibody directed against the core sequence of D-AKAP1/S-AKAP84 (14) recognized the 132-kDa band identified in the gel overlay assay. Furthermore, D-AKAP1/S-AKAP84 immunoreactivity was strongly induced after differentiation of C3H/10T1/2 cells into adipocytes (Fig. 3A) and completely abolished by preabsorption with the immunizing peptide (not shown). NPA of RNA from C3H/10T1/2 adipocytes indicated that D-AKAP1/S-AKAP84 mRNA expression is strongly induced upon adipocyte differentiation (Fig. 3B). Thus adipocyte differentiation strongly induced D-AKAP1/S-AKAP84 protein and mRNA expression.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Identification of D-AKAP1/S-AKAP84 protein and mRNA in C3H/10T1/2 adipocytes. Confluent C3H/10T1/2 cells were grown for 8-12 days in either regular medium (undifferentiated cells) or adipocyte-differentiating medium (differentiated adipocytes). A: total cellular proteins were subjected to SDS-PAGE and transferred to nitrocellulose. Blots were probed with D-AKAP1/S-AKAP84 antibody. B: detection of D-AKAP1/S-AKAP84 mRNA in undifferentiated and differentiated adipocytes by nuclease protection assay. NT, nucleotides.

Quantitative immunoblotting of recombinant RII and D-AKAP1 core peptide demonstrated that they are expressed in nearly equal amounts in differentiated adipocytes (Table 1), indicating that putative AKAP is present in amounts that might target a significant fraction of adipocyte PKA. D-AKAP1/S-AKAP84 has been localized to mitochondria in cultured cell lines and in spermatids (6, 11, 15, 18). We therefore examined the subcellular distribution of D-AKAP1 and RII in C3H/10T1/2 adipocytes by confocal microscopy. Mitochondria were visualized with the fluorescent dye MitoTracker red, whereas D-AKAP1 and RII were visualized by indirect immunofluorescence. D-AKAP1 was strongly colocalized to mitochondria, as indicated by the appearance of yellow fluorescence upon merging of the double-labeled images (Fig. 4A). In contrast, RII fluorescence showed no evidence of mitochondrial localization (Fig. 4B). Line scan analysis of confocal images demonstrated a very strong colocalization of MitoTracker red and D-AKAP1 fluorescence, whereas RII fluorescence did not localize to mitochondria (Fig. 4C). Immunocytochemical double-labeling of RII and D-AKAP1 indicated little, if any, colocalization of these proteins (Fig. 5A). Consistent with these results, line scan analysis indicated that RII and D-AKAP1/ S-AKAP84 were not significantly colocalized in these cells (Fig. 5B).

View this table: [in this window] [in a new window]   Table 1.   Quantitation of RII and D-AKAP1 in C3H/10T1/2 adipocytes

View larger version (50K): [in this window] [in a new window]   Fig. 4.   Immunolocalization of MitoTracker red, D-AKAP1/S-AKAP84, and RII in C3H/10T1/2 adipocytes. Confocal images of D-AKAP1 (A) or RII (B) are shown with Oregon green-conjugated antibodies costained with MitoTracker red, a mitochondrial fluorescent marker. Individual staining patterns as well as overlapped localization is shown. C: line scan analysis of images in A and B. D-AKAP1 (a) but not RII fluorescence (b) correlates with MitoTracker red fluorescence.

View larger version (38K): [in this window] [in a new window]   Fig. 5.   Immunolocalization of D-AKAP1/S-AKAP84 and RII in C3H/10T1/2 adipocytes. A: confocal images of D-AKAP1/S-AKAP84 with anti- D-AKAP1/S-AKAP84 antibody (red) costained with anti-RII antibody (green). Individual staining patterns as well as overlapped localization are shown. B: line scan analysis of images in A. RII fluorescence does not correlate well with that of D-AKAP1.

The above results indicate that D-AKAP1/S-AKAP84, but not RII, is targeted to mitochondria. To verify this finding with an independent biochemical technique, we subjected C3H/10T1/2 adipocyte homogenates to differential centrifugation and Percoll gradient purification of mitochondria. As shown in Fig. 6, the distribution pattern of D-AKAP1/S-AKAP84 was virtually identical to that of the mitochondrial marker prohibitin (17). Specifically, D-AKAP1/S-AKAP84 was restricted to heavy particulate fractions and highly enriched in purified mitochondria. In contrast, RII was largely excluded from the heavy particulate fraction and nearly completely absent from purified mitochondria. As a positive control for an authentic RII-interacting protein, we monitored the distribution of the C subunit of PKA and found that its distribution was identical to that of RII.

View larger version (85K): [in this window] [in a new window]   Fig. 6.   Western blot analysis of purified mitochondrial fractions obtained from C3H/10T1/2 adipocytes. Mitochondrial fractions were prepared as described in METHODS. The various fractions were subjected to immunoblotting and probed with antibodies directed against RII, C, D-AKAP1/S-AKAP84, and the mitochondrial marker prohibitin. Hom, homogenate; sup, supernatant.

We also examined whether the induction of D-AKAP1/S-AKAP84 that occurs upon adipocyte differentiation alters the subcellular distribution of RII. Homogenates of undifferentiated and differentiated C3H/10T1/2 cells were fractionated at 40,000 g into supernatant and membrane fractions (Fig. 7). In undifferentiated adipocytes, RII and C were found in the membrane fraction. D-AKAP1 was absent in undifferentiated cells, and prohibitin was expressed at low levels. Differentiation caused a marked induction of all four proteins; however, their subcellular distribution was distinct. After differentiation, both RII and C were nearly exclusively localized to the high-speed supernatant, whereas both D-AKAP1 and prohibitin were exclusively localized in the high-speed pellet. Very low levels of RII (<1% of total) could be found in membrane fractions. Because D-AKAP1 has been reported to bind both RI and RII (14), we examined the distribution of RI as well as RII in the 40,000 g supernatant and membrane fractions of differentiated C3H/10T1/2 adipocytes. Both R1 and RII immunoreactivity was found primarily in the supernatant (Fig. 8).

View larger version (72K): [in this window] [in a new window]   Fig. 7.   Western blot analysis of 40,000 g pellet and supernatant fractions from C3H/10T1/2 adipocytes. Undifferentiated and differentiated C3H/10T1/2 adipocytes were homogenized in Tris-EDTA-sucrose (TES) buffer and centrifuged at 40,000 g. The pellet and supernatant proteins were subjected to SDS-PAGE and transferred to nitrocellulose. Blots were probed with antibodies directed against RII, C, D-AKAP1/S-AKAP84, and the mitochondrial marker prohibitin.

View larger version (10K): [in this window] [in a new window]   Fig. 8.   Western blot analysis of 40,000 g pellet and supernatant fractions from C3H/10T1/2 adipocytes. Undifferentiated and differentiated C3H/10T1/2 adipocytes were homogenized in TES buffer and centrifuged at 40,000 g. The pellet and supernatant proteins were subjected to SDS-PAGE and transferred to nitrocellulose. Blots were probed with antibodies directed against RI (A) or RII (B).

To determine whether membrane-associated RII represented a weak interaction with any AKAP, we prepared C3H/10T1/2 adipocyte membranes in the presence and absence of Ht 31 peptide, which abolishes AKAP-RII interactions in vitro. Fractionation of C3H/10T1/2 adipocytes in the presence of Ht 31 peptide did not significantly alter the relative distribution of D-AKAP1 and RII in membrane fractions (Fig. 9).

View larger version (14K): [in this window] [in a new window]   Fig. 9.   Effect of Ht 31 peptide on RII and D-AKAP1 immunoreactivity in 40,000 g pellets obtained from C3H/10T1/2 adipocytes. C3H/10T1/2 adipocytes were homogenized in TES buffer, incubated in the presence or absence of Ht 31 peptide (1.5 µM), and centrifuged at 40,000 g. Pellet proteins were subjected to SDS-PAGE and transferred to nitrocellulose. Blots were probed with antibodies directed against RII and D-AKAP1. Results are expressed as the ratio of RII-to-D-AKAP1 immunoreactivity (means ± SE, n = 5).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Growing evidence suggests that PKA signaling in adipocytes is functionally compartmentalized, although the basis of that compartmentalization is unknown. One possibility is that PKA activity is directed to specific subcellular locations through interactions with targeting proteins (7). Numerous proteins that bind RII have been identified, although their role in targeting PKA in vivo is largely lacking. D-AKAP1/ S-AKAP84 was first identified on the basis of its ability to interact with RII in vitro (14, 18). Utilizing gel overlay assays, we determined that D-AKAP1/S-AKAP84 is the major RII binding protein present in brown adipose tissue and in C3H/10T1/2 adipocytes. Furthermore, D-AKAP1/S-AKAP84 protein and mRNA expression were strongly induced upon adipocyte differentiation. Quantitative immunoblotting demonstrated that D-AKAP1/S-AKAP84 and RII are present in nearly equal amounts in C3H/10T1/2 adipocytes. Given the high affinity of RII for D-AKAP1/S-AKAP84 in vitro, these observations raised the possibility that subcellular targeting of PKA might be achieved through this interaction in vivo.

Analysis of the subcellular distribution of D-AKAP1/S-AKAP84 by confocal microscopy and subcellular fractionation clearly demonstrated that D-AKAP1/S-AKAP84 is targeted to mitochondria in C3H/10T1/2 adipocytes. These observations confirm previous results demonstrating that D-AKAP1/S-AKAP84 is targeted to the outer mitochondrial membrane in spermatids and transfected HEK-293 cells (6, 11, 15, 18). The present results, however, do not support a role for D-AKAP1/S-AKAP84 in the subcellular targeting of PKA. Thus fractionation studies demonstrated that the distribution of RII correlated perfectly with that of C, and both proteins were excluded from fractions that were greatly enriched in D-AKAP1/S-AKAP84. Immunocytochemical analysis of intact adipocytes confirmed the mitochondrial targeting of D-AKAP1/S-AKAP84 in intact cells, as well as the exclusion of RII from this organelle. Although it has been reported that overexpression of D-AKAP1 in HEK-293 cells increases the association of RII with mitochondria (6), our results indicate that D-AKAP1 does not sequester RII to specific subcellular compartments in adipocytes. Indeed, adipocyte differentiation, which strongly upregulated D-AKAP1/S-AKAP84 expression in membranes, was correlated with a cytosolic distribution of PKA subunits. Very small levels of RII could be found in mitochondrial fractions, raising the possibility that D-AKAP1 might play a very modest role in mitochondrial targeting. However, this does not appear to be the case, because fractionation in the presence of Ht 31 peptide, which abolishes RII-AKAP interactions in vitro, had no effect on the low amounts of RII present in membrane fractions. Therefore, the presence of RII in membrane fractions most likely represents minor cytosolic contamination.

D-AKAP1 can bind both RI and RII subunits (14, 18), and it might be argued that D-AKAP1 plays a role in PKA signaling by tethering RI or RII subunits to specific locations inside the adipocyte. This seems unlikely, however, for several reasons. Most significantly, the catalytic subunit C of PKA, which binds all R subunits, was colocalized with RII and not with D-AKAP1. This finding is not surprising given that adipocytes express only low levels of RI or RII subunits (8). Nonetheless, the subcellular distribution of RI and RII paralleled that of the predominant RII.

Putative AKAPs have been defined by the ability of these proteins to interact with RII in artificial systems such as gel overlay and yeast two-hybrid assays (4, 11, 18, 20). This binding activity is blocked by the AKAP-inhibitory peptides such as Ht 31, indicating a common mode of interaction with RII. However, the observation that some tissues contain dozens of proteins that bind RII in vitro in a Ht 31-competitive fashion (4, 20) challenges the specificity of the overlay approach and its relevance to PKA targeting in vivo. For example, despite the high abundance of D-AKAP1/S-AKAP84 in sperm, modulation of sperm motility by Ht 31 peptide appears to be independent of both PKA targeting (1) and activity (20). The recent discovery of proteins other than RII that bind AKAPs in sperm further questions the role of AKAPs as PKA targeting proteins, at least in some tissues (2).

The expression of D-AKAP1 appears to be associated with mitochondrial function. D-AKAP1 is highly expressed in brown fat, a tissue known for its high mitochondrial content, and D-AKAP1 gene expression is strongly correlated with mitochondriogenesis that takes place upon differentiation of C3H/10T1/2 cells. The observation that D-AKAP1 does not target RII in vivo despite its high affinity in vitro suggests that the determinants of this interaction are unavailable in vivo. Future studies are necessary to determine whether these proteins have additional binding partners that confer their distinct distribution in vivo.