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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Neuroscience. Author manuscript; available in PMC Nov 24, 2011.
Published in final edited form as:
PMCID: PMC2957303
NIHMSID: NIHMS236929

Nicotine-induced up regulation of α4β 2 neuronal nicotinic receptors is mediated by the PKC-dependent phosphorylation of α4 subunits

Abstract

Sustained exposure to nicotine is well known to increase the cell surface density of α4β 2* neuronal nicotinic receptors both in vivo and in vitro, but the cellular mechanisms mediating this effect are equivocal. Using a pharmacological approach to investigate the effects of nicotine on receptor subunit expression and phosphorylation in SH-EP1 cells expressing human α4 and β 2 nicotinic receptor subunits, we have demonstrated that incubation with nicotine for 24 hours increased the expression of immature and mature forms of both α4 and β 2 subunits in a concentration-dependent manner, and that inhibition of PKC, but not PKA inhibited the nicotine-induced increased expression of subunits. Incubation of cells with nicotine for 24 hours also increased the phosphorylation of immature forms of α4 subunits similar to that induced by activation of either PKC or PKA. When cells were preincubated with nicotine, the PKC-mediated increased phosphorylation was inhibited; the PKA-mediated phosphorylation was unaltered. The phosphopeptide maps for immature α4 subunits following nicotine exposure or PKC activation were identical, and phosphoamino acid analyses indicated phosphorylation on serine residues only. Results indicate that nicotine-induced up regulation of α4β 2 neuronal nicotinic receptors involves a PKC-dependent mechanism and likely reflects the ability of nicotine to activate PKC, leading to the phosphorylation of immature α4 subunits, promoting subunit assembly and receptor maturation. Because up regulation of these receptors has been implicated to mediate tolerance, locomotor sensitization and addiction to nicotine, results identify a potential new target for modulating the effects of nicotine on the brain.

Keywords: nicotinic receptor, protein kinase A, protein kinase C, receptor phosphorylation, receptor maturation

It has been known for nearly 20 years that neuronal nicotinic receptors containing α4 and β 2 subunits exhibit an increased density following sustained exposure to nicotine (Flores et al., 1992). This is a 'universal' phenomenon that occurs in cultured neurons expressing these receptors (Pacheco et al., 2001), following injection of subunit mRNAs into Xenopus laevis oocytes (Fenster et al., 1999), and in brain from rats (Schwartz and Kellar, 1983), mice (Marks et al., 1993), and humans (Benwell et al., 1988), and is believed to be responsible for many of the effects resulting from sustained nicotine exposure including tolerance, locomotor sensitization, and the addictive process (Tapper et al., 2004). Although studies have demonstrated that sustained nicotine exposure increases the density of α4β 2* neuronal nicotinic receptors (*indicates that other subunits may be present), the mechanisms involved remain equivocal. Studies have suggested that up regulation may be due to nicotine-induced: 1) increased half-life of plasma membrane associated receptors (Peng et al., 1994; Kuryatov et al., 2005); 2) increased proportion of receptors in a high affinity state at the cell surface (Buisson and Bertrand, 2001; Nelson et al., 2003; Vallejo et al., 2005); and 3) increased receptor subunit assembly and folding, receptor maturation and/or receptor trafficking to the cell membrane (Harkness and Millar, 2002; Nashmi et al., 2003; Darsow et al., 2005; Kuryatov et al., 2005; Sallette et al., 2005; Moroni et al., 2006).

Based on results indicating that nicotine exposure, either in vitro or in vivo, did not affect subunit steady-state mRNA levels (Peng et al., 1994), initial evidence suggested that increased receptor density must be the consequence of a post-translational event. Rothhut et al. (1996) investigated the role of cAMP-dependent protein kinase (PKA) on the assembly and expression of chicken α4β 2 receptors expressed in M10 fibroblasts and demonstrated that incubation of cells with either forskolin or nicotine increased the number of cell surface receptors to a comparable extent. Further, when cells were incubated with both forskolin and nicotine, additive effects were apparent suggesting that the ability of nicotine to increase the number of cell surface receptors could not be attributed to a PKA-mediated event. Similarly, when human embryonic kidney (HEK 293) cells expressing human α4β 2 receptors were incubated with nicotine, forskolin or PMA, the latter to activate protein kinase C (PKC), an increased cell surface receptor density was apparent, but simultaneous exposure to nicotine and the kinase activators led to synergistic effects, supporting the idea that nicotine and protein kinase activation work through distinct mechanisms (Gopalakrishnan et al., 1997).

Despite evidence suggesting that the actions of nicotine differ from those of protein kinase activation, recent studies investigating α4β 2 receptor assembly and trafficking in both clonal cells and cultured midbrain neurons demonstrated that nicotine incubation increases receptor assembly and functional responses similar to that following PKC activation (Nashmi et al., 2003). Based on these findings, the authors suggested that the ability of nicotine to increase cell surface receptor density may involve a PKC-mediated mechanism involving phosphorylation at key residues on α4β 2 receptors perhaps inhibiting recognition of an ER retention sequence, and facilitating surface translocation.

The idea that phosphorylation of nicotinic receptor subunits promotes receptor assembly is supported by studies on both muscle and Torpedo nicotinic receptors demonstrating that the PKA- or PKC-mediated phosphorylation of unassembled γ or δ subunits promotes receptor assembly efficiency and increases the number of cell surface receptors (Green et al., 1991a; 1991b; Ross et al., 1991). Indeed, studies have shown that: 1) both PKA and PKC phosphorylate neuronal α4 subunits (Hsu et al., 1997; Wecker et al., 2001; Viseshakul et al., 1998; Guo and Wecker, 2002; Pacheco et al., 2003); 2) the PKA-mediated phosphorylation of α4 subunits enhances its affinity for the 14-3-3 chaperone protein, resulting in increased expression of α4β 2 receptors (Jeanclos et al., 2001); and 3) stimulation of either PKA or PKC increases α4β 2 receptor density (Pollock et al., 2009). These findings all support the possibility that the increased density of receptors induced by nicotine involves the PKA- or PKC-mediated phosphorylation of α4 subunits, enhancing the assembly and translocation of α4β 2* receptors.

To ascertain whether the nicotine-induced increased density of α4β 2 receptors could be attributed to a PKA- or PKC-dependent mechanism, human clonal epithelial cells stably transfected with human α4β 2 receptors (SH-EP1-hα4β 2 cells) were exposed to nicotine in the presence or absence of compounds that modify the activity of PKA and PKC, and receptor subunit expression and phosphorylation were determined.

EXPERIMENTAL PROCEDURES

Materials

The following antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were used for immunoblotting: a primary polyclonal anti-α4 antibody (H-133, sc-5591); a primary polyclonal anti-β 2 antibody (H-92, sc-11372); a secondary goat anti-rabbit-HRP antibody (sc-2030); a goat anti-human actin primary antibody (C-11, sc-1615); and a donkey anti-goat-HRP secondary antibody (sc-2033). For immunoprecipitation experiments, the anti-α4 monoclonal antibody mAb299 was used (MRT-613R, Covance Research Products, Denver, PA) as it has been shown that this antibody binds to both the lower and higher molecular weight species of the α4 subunit (Pollock et al., 2007; 2009). 32Pi was purchased from Perkin-Elmer Life and Analytical Sciences Inc. (Boston, MA), X-ray film from Eastman Kodak Co. (Rochester, NY), enhanced chemiluminescence (ECL) reagents from Amersham-Biosciences Corporation (Piscataway, NJ), and polyvinylidene difluoride (PVDF) membranes (Immobilon-P) from the Millipore Corporation (Billerica, MA). DMEM, penicillin, streptomycin, L-glutamine, and horse serum were purchased from Invitrogen (Carlsbad, CA), Fetal Clone II from HyClone (Logan, UT), and forskolin, PDBu and RO-31-8220 from Calbiochem (San Diego, CA). Basic electrophoresis chemicals and protein markers were obtained from Bio-Rad Laboratories (Hercules, CA), and protein G and additional molecular weight markers were purchased from Sigma-Aldrich (St. Louis, MO). Thin-layer chromatography (TLC) plates were obtained from VWR International Inc. (Bristol, CT) and sequencing grade trypsin was purchased from Roche Diagnostics Corporation (Indianapolis, IN).

Expression of mature and immature α4 and β 2 subunit proteins

Human clonal SH-EP1-hα4β 2 cells (kindly provided by Dr. Ron Lukas, Barrow Neurological Institute, Phoenix, AZ) were used for all studies. Cells were grown and maintained at 37°C (with 5% CO2) in DMEM (high glucose) containing 5% fetal clone II, 10% horse serum, 1 mM sodium pyruvate, 8 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.05 mg/ml amphotericin B, 0.5 mg/ml zeocin and 0.4 mg/ml hygromycin B, the latter two for selection of the receptor. Cells were kept in continuous culture, fed every 2–3 days, and were grown to 80–85% confluence in 60 mm dishes.

For studies investigating the concentration-dependent effects of nicotine on the expression of α4 and β 2 subunits, cells were washed and incubated with nicotine (0–1000 nM) for 24 hours. For studies investigating the effects of PKC inhibition on the up regulation of α4 and β 2 subunit protein, cells were incubated in the absence or presence of the PKC inhibitor RO-31-8220 (0.1 μM) for 30 minutes, followed by the addition of forskolin (10 mM in 0.1% DMSO), phorbol 12,13-dibutyrate (PDBu, 200 nM in 0.1% DMSO), or nicotine (50 nM) and incubated for 24 hours. Following incubation, whole cell lysates were prepared by washing the cells (5 x) with ice-cold phosphate buffered saline (PBS) followed by incubation for 30 minutes at 4°C in 400 ml of potassium phosphate (200 mM) homogenization/lysis buffer (pH 7.4) containing 150 mM NaCl, 10 mM EDTA, 10 mM EGTA, 10 mM β -glycerophosphate, 50 mM NaF, 1 mM Na3VO4, 0.1 mM phenylmethylsulfonyl fluoride, 1 mg/ml pepstatin A, 10 μg/ml leupeptin, 10 U/ml aprotinin, and 2% Triton X-100. The plates were scraped and samples were transferred into microfuge tubes and triturated by mechanical disruption (20 strokes) using a 1 ml syringe with a 26-gauge needle; samples were further solubilized for 30 minutes at 4°C on a rotator. Samples were centrifuged at 20,000 × g for 20 minutes, and the supernatants were withdrawn and stored at −80°C. For immunoblotting, samples were thawed on ice, resuspended in an equal volume of 2X Laemmli buffer (Laemmli, 1970) and boiled for 5 minutes. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 7.5% acrylamide gels and transferred to PVDF membranes. The membranes were blocked with Tris-buffered saline (TBS) containing 5% non-fat dry milk and 0.05% Tween-20 for 2 hours at 24°C. To determine the relative quantities of both α4 and β 2 subunit protein, the membranes were cut horizontally just above the 59 kDa prestained marker between the α4 and β 2 protein bands, and the top halves of the membranes were probed for α4 subunit protein and the bottom halves for β 2 subunit protein. The relative quantity of α4 subunit protein was determined using a rabbit polyclonal antibody raised against a recombinant protein corresponding to amino acids 342–474 of the human α4 subunit (H-133, 200 mg/ml used at a 1:2000 dilution); β 2 subunit protein was determined using a rabbit polyclonal antibody raised against a recombinant protein corresponding to amino acids 342–433 of the human β 2 subunit (H-92, 200 mg/ml used at a 1:2000 dilution). Membranes were incubated with their respective antibodies in TBS containing 5% non-fat dry milk and 0.05% Tween-20 for 1 hour at 24°C. The membranes were washed and incubated with horseradish peroxidase conjugated goat anti-rabbit secondary antibody (400 μg/ml used at 1:2000 dilution) for 30 minutes, and the α4 and β 2 signals were visualized using ECL. Blots were also probed for β -actin to normalize among samples. Immunoblots were analyzed by densitometry using a Bio-Rad Imaging Densitometer with Multi-Analyst software (Bio-Rad Laboratories, Hercules, CA). Sigma “Precision Plus” Kaleidoscope protein markers were used for reference.

32Pi labeling and α4 subunit phosphorylation

SH-EP1-hα4β 2 cells were grown and maintained to 80–85% confluence as above. To label endogenous ATP stores, the cells were washed 3 times with 2 ml phosphate-free DMEM containing 8 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin, and incubated at 37°C in 5% CO2 for 4 hr in phosphate-free media containing 0.5 mCi/ml 32Pi (1 mCi/plate for phosphorylation experiments and 2 mCi/plate for phosphopeptide mapping).

For experiments investigating the effects of nicotine on α4 subunit phosphorylation, cells were incubated in DMEM in the absence or presence of 50 nM nicotine for 20 hours prior to the 4 hour labeling period. Following the 20 hour incubation period in the absence of 32Pi, cells were washed and incubated with 32Pi as above for 4 hours in the absence or presence of 50 nM nicotine, the latter to control for any potential reversible nicotine-induced alterations during this time. For experiments investigating the effects of nicotine on the PKA- and PKC-dependent phosphorylation of α4 subunits, cells were incubated in the absence or presence of 50 nM nicotine for 24 hours in the absence of 32Pi, washed, and incubated with 32Pi for 4 hours. Forskolin (10 μM) or PDBu (100 nM) in 0.1% DMSO was added during the final 15 or 30 minutes of the 4 hour labeling period, respectively. These concentrations and times of incubation were chosen based on studies indicating that these parameters led to maximal kinase activation and did not alter the amount of 32Pi taken up by the cells during the 4 hour labeling period.

Following incubation, cells were washed 5× with ice-cold phosphate buffered saline (PBS), followed by the addition of 500 μl aliquots of homogenization buffer (200 mM potassium phosphate, 150 mM NaCl, 10 mM EDTA, 10 mM EGTA, 10 mM β -glycerophosphate, 50 mM NaF, 1 mM NaVO3, 0.1 mM phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A, 10 μg/ml leupeptin, and 10 U/ml aprotinin). The plates were scraped, the cells were transferred to 1.5 ml centrifuge tubes and triturated by mechanical disruption as above. Samples were centrifuged at 500 × g for 5 min at 4°C, and the supernatants were withdrawn and centrifuged at 20,000 × g for 30 min at 4°C. The resultant membrane pellets were resuspended in homogenization buffer containing 2% Triton X-100, solubilized for 30 min at 4°C, and centrifuged at 230,000 × g for 10 min at 4°C. The supernatants containing the 32P-labeled solubilized proteins were withdrawn and α4 subunit protein was immunoprecipitated from the detergent extracts by rotating overnight at 4°C with 5 μg of mAb 299 pre-coupled to protein-G sepharose beads. This monoclonal antibody preparation has been shown to bind to both the predominant immature 71–75 kDa α4 protein, as well as the mature 80–85 kDa protein (Pollock et al., 2007; 2009). The bead/antibody/receptor complex was washed 5 times with 100 ml of final storage buffer (50 mM MOPS, 1 mM Na2EDTA, 1 mM EGTA, pH 7.2 and 0.2% Triton-X 100), resuspended in 20 μl Laemmli buffer and boiled for 5 minutes. Following centrifugation at 2,000 × g for 2 minutes, the proteins from the supernatant were separated on SDS-PAGE gels and transferred to PVDF membranes. The membranes were exposed to Kodak XAR film in the presence of intensifying screens for 2–5 hours at −80°C to visualize 32P-labeled α4 subunit protein by autoradiography prior to immunoblotting. The relative quantity of α4 subunit protein was determined by immunoblot analysis using H-133 as above. Autoradiographs and immunoblots were quantitated by densitometry and the relative phosphorylation was expressed as the ratio of the 32P signal from the autoradiograph divided by the protein level from the immunoblot.

Phosphopeptide mapping and phosphoamino acid analyses

Two-dimensional (2-D) phosphopeptide mapping and phosphoamino acid analysis of the immunoprecipitated 32P-labeled α4 subunits were performed using standard techniques as described in detail (Pollock et al., 2007; 2009). Briefly, PVDF membrane bands containing the 32P-labeled α4 protein were excised and processed for either 2-D phosphopeptide mapping or phosphoamino acid analysis. For the former, samples were digested with trypsin, spotted onto cellulose TLC plates, and subjected to electrophoresis in the first dimension and ascending chromatography in the second dimension. Tryptic phosphopeptide patterns were visualized by exposing the plates to XAR film with intensifying screens. Due to large differences in the amount of phosphorylated material obtained under different experimental conditions, within each set of experiments, samples containing equal amounts of radioactivity (1000 cpm) and not equal amounts of protein were analyzed.

For phosphoamino acid analysis, samples were digested with acid and resuspended in buffer containing the standards phosphoserine (S), phosphothreonine (T) and phosphotyrosine Y). Samples containing 100 cpm were spotted onto cellulose TLC plates, and were subjected to electrophoresis at 1.5 kV for 20 min in pH 1.9 buffer in the horizontal direction, and at 1.6 kV for 16 min in pH 3.5 buffer in the vertical dimension. The migration of the phosphoamino acid standards was visualized by spraying the TLC plates with 0.25% (w/v) ninhydrin followed by exposing the plates to Kodak XAR film with intensifying screens for 1–6 weeks at −80°C. The kinase-dependent phosphorylation of serine, threonine, or tyrosine was identified based on the co-migration of the 32P-labeled residues with the stained phosphoamino acid standards.

Statistical Analyses

Grouped data are presented as the mean + S.E.M. For concentration-response curves, data were analyzed using GraphPad PRISM (San Diego, CA). For all other experiments, data were analyzed by analysis of variance (ANOVA); in those instances where significant (p < 0.05) main effects were noted, individual group differences were determined by Newman-Keuls test. A level of p < 0.05 was accepted as evidence of a statistically significant effect.

RESULTS

Nicotine increases receptor subunit expression by a PKC-dependent mechanism

The first series of experiments characterized the concentration-dependent effects of nicotine on the expression of α4 and β 2 subunits associated with immature (α4i and β 2i) and mature (α4m and β 2m) complexes, the former representing subunits present in complexes located primarily in the endoplasmic reticulum, and the latter representing a mixture of high mannose and complex oligosaccharides likely corresponding to subunits isolated from fully processed mature forms of the receptor associated with the trans-Golgi and plasma membrane (Sallette et al., 2005; Pollock et al., 2009). Results demonstrate that both immature and mature species of α4 and β 2 were apparent in the cell lysates (Figure 1a) with α4i (71–75 kDa) and β 2i (47 kDa) predominant and representing approximately 3–4 times the amount of α4m (80–85 kDa) and β 2m (50 kDa) when normalized to β -actin (Figure 1b), in agreement with prior studies (Harkness and Millar, 2002; Sallette et al., 2005; Pollock et al., 2007). Following incubation of cells with nicotine for 24 hours, concentration-dependent increases in the amount of both species of α4 and β 2 subunits were apparent with a maximal effect following 24 hours incubation with 300 nM nicotine. When data were expressed as percent of corresponding controls, significant (p<0.05) increases were apparent at all concentrations of nicotine tested for all subunit species (Figure 1c). Further, at the lowest concentrations of nicotine (50 and 100 nM), the relative increase in mature and immature forms of α4 subunits did not differ from each other, but as the concentration of nicotine increased, a greater effect was apparent on the expression of the mature form of α4, with a significant (p<0.05) increase in the expression of α4m relative to α4i following incubation with 300 and 1000 nM nicotine, consistent with the action of nicotine as a maturation enhancer, stabilizing α4β 2 complexes and increasing their surface expression (Sallette et al., 2005).

Fig. 1
Effects of nicotine on the expression of α4 and β 2 subunit protein. SH-EP1-hα4β 2 cells were incubated with 0–1000 nM nicotine for 24 hours. Whole cell lysates were prepared, proteins were separated by SDA-PAGE ...

Based on data suggesting that the ability of nicotine to increase cell surface density may involve the PKC-dependent phosphorylation of α4β 2 receptors facilitating surface translocation (Nashmi et al., 2003), experiments investigated whether inhibition of PKC could prevent the nicotine-induced up regulation. To accomplish this, cells were incubated for 24 hours with 10 mM forskolin, 200 nM PDBu or 50 nM nicotine, conditions that have been shown to increase the number of membrane-associated α4β 2 receptors in these cells (Pacheco et al., 2003; Pollock et al., 2009), in the absence or presence of the PKC inhibitor RO-31-8220 (0.1 μM). Following incubation, whole cell lysates were prepared, proteins were separated, and immunoblots were probed for the expression of the mature and immature species of both α4 and β 2 subunit proteins. A representative immunoblot and grouped data are shown in Figure 2. Results indicate that following incubation of cells with forskolin, PDBu or nicotine, the relative amounts of both species of α4 and β 2 subunits increased significantly (p<0.05). Further, the presence of the PKC inhibitor RO-31-8220 totally prevented the nicotine- and PDBu-induced increases in the immature and mature forms of both α4 and β 2 subunits. The specificity of RO-31-8220 as a PKC inhibitor was confirmed by evidence that this compound had no effect on foskolin-induced increases. Further, incubation with RO-31-8220 by itself had no effect on subunit abundance. These results support the idea that the ability of nicotine to up regulate α4β 2 receptors is mediated through a PKC-dependent process, perhaps involving the phosphorylation of α4 subunit

Fig. 2
Effects of nicotine and protein kinase activation and inhibition on the expression of α4 and β 2 subunit protein. SH-EP1-hα4β 2 cells were incubated in the absence or presence of the PKC inhibitor RO-31-8220 (0.1 μM) ...

Nicotine phosphorylates α4i subunit expression by a PKC-dependent mechanism

To investigate the possibility that the effects of nicotine are mediated by PKC-dependent phosphorylation, the effects of nicotine on the phosphorylation of α4 subunits were investigated. To accomplish this, cells were incubated in the absence or presence of 50 nM nicotine for 20 hours prior to 4 hours incubation with 32Pi to label ATP stores. To control for the possibility that α4 subunits were phosphorylated during the 20 hour incubation with nicotine, followed by dephosphorylation during the 4 hour labeling period, the labeling incubation was conducted both in the presence and absence of 50 nM nicotine. Following incubation, α4 subunits were immunoprecipitated from a membrane preparation, and subunit phosphorylation of the most abundant α4 species (α4i) was determined. A representative autoradiograph and immunoblot and grouped data are shown in Figure 3. Results indicate that incubation of cells with 50 nM nicotine for 20 hours led to an 88% increase (p<0.05) in the phosphorylation of α4i (condition [C]). Results also demonstrate that the presence of nicotine only during the 4 hour labeling period (condition [B]) led to a 23% increase (p<0.05) in subunit labeling, and that when nicotine was present during both the 20 hour incubation and the 4 hour labeling period (condition [D]), the increased phosphorylation of α4i did not differ significantly from that achieved when nicotine was absent during the labeling period. Thus, results indicate that prolonged exposure of cells to 50 nM nicotine leads to increased α4i subunit phosphorylation in a time-dependent manner. Further, because the increased phosphorylation following 20–24 hours incubation of cells with nicotine was significantly (p<0.05) greater than the increase achieved when nicotine was present only during the labeling period, this effect of nicotine could not be attributed to an action of nicotine to increase 32Pi uptake by cells.

Fig. 3
Effects of nicotine on the phosphorylation of α4i subunits. SH-EP1-hα4β 2 cells were incubated in the absence or presence of 50 nM nicotine for 20 hours, washed, and incubated for an additional 4 hours with 32Pi in the absence ...

Based on studies indicating that nicotine activates PKC (Messing et al., 1989; Tuominen et al., 1992; Koide et al., 2005), in concert with evidence of increased activity of PKA in postmortem brain from cigarette smokers (Hope et al., 2007), it was possible that the nicotine-induced increased α4i subunit phosphorylation involved activation of one or both of these kinases. Indeed, studies in our laboratory have demonstrated that activation of PKA by forskolin or PKC by PDBu increases α4 subunit phosphorylation (Pacheco et al., 2003; Pollock et al., 2007; 2009). Thus, to determine whether the ability of nicotine to enhance α4 subunit phosphorylation was due to the activation of PKA or PKC, cells were incubated with 50 nM nicotine for 24 hours in the absence of 32Pi, washed and incubated for 4 hours with 32Pi. Forskolin (10 μM) or PDBu (200 nM) were added during the final 15 or 30 minutes of the labeling period, respectively [concentrations and times of incubation that produce maximal kinase activation without altering the total radioactivity taken up by cells during the 4 hour labeling period and lead to increased α4i subunit phosphorylation (Pacheco et al., 2003; Pollock et al., 2007; 2009)], and the phosphorylation of both the mature and immature species of the α4 subunit was determined. It was predicted that if nicotine phosphorylated α4 by activation of either or both of the kinases, then following exposure of the cells to nicotine, incubation with forskolin or PDBu should be without effect. A representative autoradiograph and immunoblot and grouped data are shown in Figure 4. In agreement with prior studies (Pollock et al., 2009), results indicate that both forskolin and PDBu increased the relative phosphorylation of the immature species of α4 subunits significantly (p<0.05), and only PDBu increased phosphorylation of the mature species (p<0.05). Further, when cells were incubated with 50 nM of nicotine for 24 hours prior to exposure to the kinase activators, the PDBu-induced phosphorylation of α4i was prevented, and phosphorylation did not differ from samples incubated with nicotine alone. There was no effect of nicotine incubation on the forskolin-induced phosphorylation of α4i. In addition, nicotine incubation did not affect the phosphorylation of the mature species of α4, and did not alter the PDBu-induced phosphorylation of this form of the subunit. These results indicate that nicotine and PDBu share a common mechanism to activate PKC, leading to the phosphorylation of specific substrate sites on α4i.

Fig. 4
Effects of nicotine and protein kinase activation on the phosphorylation of α4i and α4m subunits. SH-EP1-hα4β 2 cells were incubated in the absence (open bars) or presence (hatched bars) of 50 nM nicotine for 24 hours in ...

To test this possibility, cells were labeled with 32Pi for 4 hours in absence or presence of 50 nM nicotine or 200 nM PDBu during the final 30 minutes of incubation. Proteins were immunoprecipitated from detergent-solubilized membranes with mAb299 and separated by SDS-PAGE gel electrophoresis. The labeled immature α4 subunit protein was excised from the membranes, digested with trypsin and subjected to 2-D phosphopeptide mapping and phosphoamino acid analysis. Results (Figure 5) indicate that samples incubated with nicotine yielded 2-D phosphopeptide maps for α4i that were identical to those following incubation with PDBu. Of particular importance is the increased phosphorylation of phosphopeptide fragments contained within clusters C5 and C6, which represent residues phosphorylated only on immature α4 subunits following stimulation of PKC (Pollock et al., 2009). It is also important to note that under control conditions, peptide fragments contained within cluster C5 were not phosphorylated appreciably and those within C6 were not phosphorylated at all. Further, these fragments are phosphorylated only on immature and not mature α4 subunits, and are not phosphorylated upon stimulation of PKA with forskolin (Pollock et al., 2007; 2009). Phosphoamino acid analysis indicates that only serine residues are phosphorylated under any condition examined. These data indicate that nicotine and PKC phosphorylate two common sites on immature α4 subunits and strongly suggest that nicotine may have its actions by stimulating PKC.

Fig. 5
Two-dimensional phosphopeptide maps and phosphoamino acid analyses of α4i following incubation of cells with nicotine or PDBu. SH-EP1-hα4β 2 cells were labeled with 32Pi and incubated in the absence or presence of 50 nM nicotine ...

DISCUSSION

The objective of the current experiments was to determine whether the well documented nicotine-induced up regulation of α4β 2 receptors was due to a PKA- or PKC-dependent mechanism involving the phosphorylation of α4 neuronal nicotinic receptor subunits. Results indicate that: 1) incubation of SH-EP1-hα4β 2 cells with nicotine for 24 hours led to a concentration-dependent increase in the expression of immature and mature forms of both α4 and β 2 subunits similar to that induced by incubation of the cells with either forskolin or PDBu to activate PKA or PKC, respectively; 2) the nicotine- and PDBu-induced increased expression of α4 and β 2 subunits was prevented by concurrent incubation with the PKC inhibitor RO-31-8220; 3) incubation of cells with nicotine for 24 hours led to increased phosphorylation of immature forms of α4 subunits similar, albeit not as robust as that induced by incubation of the cells with either forskolin or PDBu; 4) incubation of cells with nicotine for 24 hours prevented the PDBu-induced phosphorylation of immature, but not mature α4 subunits and did not affect the forskolin-induced phosphorylation of the former; and 5) both nicotine and PDBu phosphorylate only serine residues on immature α4 subunits and lead to the generation of virtually identical phosphopeptide fragments from α4i. These results indicate that nicotine-induced up regulation of α4β 2 neuronal nicotinic receptors involves a PKC-dependent mechanism and likely reflects the ability of nicotine to activate PKC, leading to the phosphorylation of immature α4 subunits, enhancing neuronal nicotinic receptor subunit assembly and receptor maturation.

More than 20 years ago, TerBush and Holz (1986) reported that nicotinic agonists activated PKC in bovine adrenal chromaffin cells, and since that time, studies have replicated this finding (Tuominen et al., 1992), and have shown that nicotinic receptor stimulation increases the translocation/activation of PKC using several cell types including PC12 cells (Messing et al., 1989), smooth muscle cells (Koide et al., 2005), and human umbilical vein endothelial cells (Ueno et al., 2006). This finding can now be extended to include SH-EP1-hα4β 2 cells as evidenced by: 1) the generation of identical 2-D phosphopeptide maps and the enhanced phosphorylation of two common sites on immature α4 subunits following incubation of cells with either nicotine or PDBu; 2) inhibition of nicotine- and PDBu-induced up regulation of α4 and β 2 subunits by the PKC inhibitor RO-31-8220; and 3) prevention of PDBu-induced α4 subunit phosphorylation by prior exposure of cells to nicotine. Measures of PKC activity in cells (data not shown) indicated that nicotine did not activate PKC directly. It is likely that nicotine, by activating cell surface receptors, increases intracellular Ca2+ concentrations, decreasing the affinity of the pseudosubstrate domain for the PKC catalytic site leading to protein unfolding and enzyme activation, directing the enzyme to the endoplasmic reticulum via the RACK anchoring protein. This idea is consistent with evidence that nicotine has a 'maturating enhancing' effect on immature α4β 2 complexes and may 'rescue' misfolded oligomers that otherwise may be degraded (Sallette et al., 2005), perhaps by stabilizing conformations through PKC-mediated subunit phosphorylation. It is also possible that this mechanism represents the previously undefined 'second messenger signaling system' whereby the activation of cell surface receptors by nicotine increases the number of receptors transiting from the endoplasmic reticulum to the cell membrane, as suggested by Darsow et al. (2005).

Until recently, most studies using immunoblot analyses to investigate alterations in the ability of nicotine to increase the density of α4β 2* receptors have focused on the predominant lower molecular weight form of the α4 subunit. Sallette et al. (2005) demonstrated that both α4 and β 2 subunits exist in at least two forms that migrate as different molecular weight proteins on Western immunoblots, a more abundant lower molecular weight form representing immature species present in the endoplasmic reticulum, and a less abundant, higher molecular weight form representing mature species complexed with high mannose and oligosaccharides and associated primarily in the plasma membrane. Further, these authors demonstrated that nicotine acts intracellularly to enhance subunit complex oligomerization. Studies in our laboratory (Pollock et al., 2009) confirmed and extended these findings and identified 3 forms of the α4 subunit upon immunoprecipitation with mAb290, a high molecular weight species at 80–85 kDa, a predominant lower species at 70–75 kDa, and a minor protein band at 66 kDa, the former two corresponding to the mature and immature species and the latter likely corresponding to a fully deglycosylated degradation product as identified by Sallette et al. (2005). Further, we demonstrated that the mature species of α4 was not demonstrable in samples immunoprecipitated with mAb299 from Sigma-Aldrich Co., but was present in preparations immunoprecipitated with mAb299 obtained from Covance Inc. (Pollock et al., 2007; 2009). Results from the present study demonstrate that both species of α4 subunit protein can be detected in whole cell lysates (Figure 1), and immunoprecipitation is not necessary. Thus, using whole cell lysates for measures of subunit protein abundance eliminates apparent discrepancies between measures of receptor densities by radioligand binding versus immunoprecipitation using conformation-dependent antibodies and cell surface biotinylation studies (Kuryatov et al., 2005).

Based on evidence that incubation of cells with nicotine for 24 hours increased the expression of mature forms of the subunits to a greater extent than immature forms, results suggest that nicotine promotes maturation of the receptor complex. Further, within the limitations of these experiments, no significant changes in the ratio of α4m:β 2m were observed following nicotine exposure suggesting that nicotine did not alter the stoichiometry of mature receptors. Thus, results do not support the idea that nicotine shifts the equilibrium between high- and low-affinity states of the receptor or that nicotine promotes an expression/maturation pathway to favor one particular stoichiometry over the other, i.e., high affinity (α4)2(β 2)3 versus low affinity (α4)3(β 2)2 receptors (Nelson et al., 2003; Vallejo et al., 2005). Rather, results support the idea that nicotine acts on an immature population of subunits/oligomers to facilitate the conversion of these precursors into mature species with higher metabolic stability, leading to up regulation of the mature receptor (Sallette et al. 2005). Further, evidence supports the idea that this process is mediated by activation of PKC, as suggested but not investigated directly by Nashmi et al. (2003).

It is tempting to speculate that activation of PKC by nicotine and phosphorylation of α4 subunits promotes subunit or oligomeric complex stability by promoting interaction with chaperone proteins. Studies have demonstrated that phosphorylation of unassembled α4 subunits by PKA increases association with the 14-3-3 chaperone protein, increasing subunit steady-state levels (Jeanclos et al., 2001). The idea of a similar process involving the PKC-mediated phosphorylation of α4 subunits either in the free form or as immature α4β 2 complexes has not yet been investigated. Although the assembly and maturation of many types of nicotinic receptors depends on interactions with endoplasmic reticulum chaperone proteins (Millar and Harkness, 2008), little is known about the role of phosphorylation in mediating these protein:protein interactions. One protein of interest is the RIC-3 chaperone protein as recent studies have demonstrated a positive correlation between the expression of this chaperone protein and that of α4 and β 2 subunits in human brain (Severance and Yolken et al., 2007) and evidence that RIC-3 enhances the expression of α4β 2 receptors in human kidney tsA201 cells (Lansdell et al., 2005).

In sum, results support the idea that nicotine acts as a pharmacological chaperone (Kuryatov et al., 2005) to increase the assembly and maturation of α4β 2* neuronal nicotinic receptors, and indicate that its action is mediated by activation of PKC, resulting in the phosphorylation of immature α4 subunits on serine residues and promoting receptor assembly and maturation. Further, because PKC phosphorylation sites on α4 subunits are located within the M3/M4 cytoplasmic domain of the protein (Wecker et al., 2001; Pollock et al., 2007), results support evidence that this large intracellular loop has a major influence on receptor assembly and ultimately, function. Additional studies will undoubtedly delineate the role of α4 subunit phosphorylation in mediating protein:protein interactions required for the nicotine-induced increased expression of cell surface α4β 2* receptors, identifying new targets for the pharmacological modulation of these receptors, perhaps leading to new approaches to the treatment of addiction.

Acknowledgments

The authors gratefully appreciate Ms. Melanie Engberg for providing excellent editorial assistance. These studies were supported in part by a grant from the National Institute of Drug Abuse #DA14010 (to L.W.).

Abbreviations

α4i
immature α4 subunits
α4m
mature α4 subunits
β 2i
immature β 2 subunits
β 2m
mature β 2 subunits
ECL
enhanced chemiluminescence
HRP
horseradish peroxidase
PBS
phosphate buffered saline
PDBu
phorbol 12,13-dibutyrate
PVDF
polyvinylidene difluoride
Pi
inorganic phosphate
PKA
cAMP-dependent protein kinase
PKC
protein kinase C
SDS-PAGE
sodium dodecylsulfate polyacrylamide gel electrophoresis
TBS
Tris-buffered saline
TLC
thin-layer chromatography
2-D
two-dimensional

Footnotes

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