Phosphorylation of the α4 subunit of human α4β2 nicotinic receptors: role of cAMP-dependent protein kinase (PKA) and protein kinase C (PKC)

Molecular Brain Research 114 (2003) 65–72 www.elsevier.com / locate / molbrainres

Research report

Phosphorylation of the a4 subunit of human a4b2 nicotinic receptors: role of cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) Mary A. Pacheco, Tina E. Pastoor, Lynn Wecker* ,1 Department of Pharmacology and Therapeutics, University of South Florida College of Medicine, 12901 Bruce B. Downs Blvd., Tampa, FL 33612 -4799, USA Accepted 1 April 2003

Abstract This study determined whether the a4 subunit of human a4b2 neuronal nicotinic receptors is phosphorylated in situ by cyclic AMP-dependent protein kinase (PKA) or protein kinase C (PKC). To accomplish this, human cloned epithelial cells stably transfected with the human a4b2 nicotinic receptor (SH-EP1-ha4b2) were incubated with 32 P-orthophosphate to label endogenous ATP stores, and the phosphorylation of a4 subunits was determined in the absence or presence of PKA or PKC activation. Autoradiographs and immunoblots indicated that a4 subunits immunoprecipitated from a membrane preparation of SH-EP1-ha4b2 cells exhibited a single 32 P-labeled band corresponding to the a4 subunit protein; no signals were associated with untransfected SH-EP1 cells. The a4 subunits from SH-EP1-ha4b2 cells incubated in the absence of the activators exhibited a basal level of phosphorylation that was decreased in the presence of the PKA inhibitor H-89 (5 mM), but unaltered in the presence of the PKC inhibitor Ro-31-8220 (0.1 mM). Activation of PKA by forskolin (10 mM), dibutyryl-cAMP (1 mM), or Sp-8-Br-cAMP (1 mM) enhanced phosphorylation nearly threefold; the inactive isomer, Rp-8-Br-cAMP (1 mM) had no effect. In addition, the forskolin effect was totally blocked by the PKA inhibitor H-89 (5 mM). Activation of PKC by the phorbol esters PDBu (200 nM) or PMA (200 nM) increased a4 subunit phosphorylation approximately twofold, and the PDBu effect was blocked by the selective PKC inhibitor Ro-31-8220 (0.1 mM). These findings indicate that the a4 subunit of human a4b2 nicotinic receptors is phosphorylated in situ by PKA and PKC.  2003 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Acetylcholine receptors: nicotinic Keywords: Phosphorylation; Cyclic AMP-dependent protein kinase; Protein kinase C; Human a4b2 neuronal nicotinic receptors

1. Introduction During the past 10 years, many studies have suggested that a4b2 neuronal nicotinic receptors, the most abundant nicotinic receptor type in the brain, are regulated by

*Corresponding author. Department of Pharmacology and Therapeutics, MDC Box 09, University of South Florida College of Medicine, 12901 Bruce B. Downs Blvd., Tampa, FL 33612, USA. Tel.: 11-813974-3823; fax: 11-813-974-3081. E-mail address: [email protected] (L. Wecker). 1 http: / / hsc.usf.edu / PHARM / lw.htm.

phosphorylation / dephosphorylation mechanisms. Studies investigating chicken a4b2 receptors expressed in mouse fibroblast (M10) cells [22], human a4b2 receptors expressed in human embryonic kidney (HEK 293) cells [5,8], and rat a4b2 receptors expressed in Xenopus oocytes [6] have shown that activators and inhibitors of cyclic AMPdependent protein kinase (PKA) or protein kinase C (PKC) modify the surface expression of receptors, and alter the extent of receptor desensitization / inactivation and recovery following sustained agonist exposure. Although evidence supports a role for these kinases in modulating the expression and function of a4b2 receptors, the specific mechanisms involved and the protein substrate(s) for phosphorylation have not been determined.

0169-328X / 03 / $ – see front matter  2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0169-328X(03)00138-4

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Evidence has indicated that rat a4 neuronal nicotinic receptor subunits may be phosphorylated by either PKA or PKC. Studies on rat a4b2 receptors either isolated from brain [17,18] or expressed in and immunoprecipitated from Xenopus oocytes [11] provided initial evidence for phosphorylation of the a4 subunit by PKA. Additional evidence [28] has suggested that: (1) immunoprecipitated rat a4 subunits are a substrate for both PKA and PKC; (2) phosphorylation occurs on a fusion protein corresponding to the major cytoplasmic loop between the third (M3) and fourth (M4) transmembrane domains (M3 / M4) of the subunit; and (3) both PKA and PKC phosphorylate serine residues, but only PKC phosphorylates threonine residues. Furthermore, studies have demonstrated the 32 P-labeling of a4 subunits following incubation of Xenopus oocytes expressing rat a4b2 receptors with 32 P-orthophosphate [9,27]. Thus, both direct and indirect evidence indicate that the rat a4 neuronal nicotinic receptor subunit is a substrate for PKA and PKC. However, it is unclear whether the human a4b2 receptor is regulated in a manner analogous to the rat receptor, as studies have shown that the former exhibit sensitization following sustained agonist exposure [2,8], whereas the latter exhibit inactivation [6,10]. A recent study investigating the relative affinities of peptides corresponding to putative phosphorylation sites in the M3 / M4 cytoplasmic domain of the human a4 subunit suggests that the human a4 subunit may be a substrate for PKA or PKC [29], but there is no direct evidence to support phosphorylation of the human a4 subunit. Analysis of the deduced amino acid sequence of the a4 protein from rat and human has indicated that the M3 / M4 cytoplasmic loop contains more than 20 putative amino acid consensus sequences for serine / threonine protein kinases [1]. Of these sites, although several sequences are highly conserved between rat and human, many of these are not homologous, and in a few instances, one species contains a threonine within the consensus sequence while the other contains a serine. PKA has been shown to phosphorylate only serines while PKC phosphorylates both serines and threonines on the rat a4 subunit [28], thus, the specific amino acid present could be a primary determinant for kinase specificity. This study determined whether a4 subunits of human a4b2 receptors are phosphorylated, and if so, whether PKA or PKC are involved. To accomplish this, human clonal epithelial cells stably transfected with human a4b2 receptors (SH-EP1-ha4b2 cells) were incubated with 32 Porthophosphate, and the phosphorylation of a4 subunits was determined in the absence or presence of PKA or PKC activation. These cells have been shown to represent a good model system to study human a4b2 receptors because they express a single class of high affinity binding sites for agonist and respond to nicotine in a manner analogous to the rat receptor expressed in Xenopus oocytes [7,20].

2. Materials and methods

2.1. Materials 32

P-Orthophosphate (285.5 Ci / mg; 150 mCi / ml) was purchased from Perkin-Elmer Life Sciences (Boston, MA, USA). The primary anti-a4 antibody (SC5591) and the secondary horseradish peroxidase-conjugated anti-rabbit antibody used for immunoblotting were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA, USA); the anti-a4 antibody used for immunoprecipitation (mAb299) was purchased from Covance (Richmond, CA, USA). XRay film was purchased from Eastman Kodak (Rochester, NY, USA), the enhanced chemiluminescence reagents were obtained from Amersham Biosciences (Piscataway, NJ, USA), and polyvinylidene fluoride (PVDF) microporous membranes (Immobilon-P) were bought from Millipore (Bedford, MA, USA). DMEM, penicillin, streptomycin, L-glutamine, and horse serum were purchased from Invitrogen (Carlsbad, CA, USA), and Fetal Clone II from Hyclone Laboratories (Logan, UT, USA). Protein kinase activators [forskolin, Sp-8-Br-cAMP, dibutyryl cAMP (dbcAMP), PDBu and PMA], the inactive isomer Rp-8-BrcAMP, and the inhibitors (H-89 and Ro-31-8220) were purchased from Calbiochem (San Diego, CA, USA). All other reagents were from Sigma (St. Louis, MO, USA).

2.2. 32 P-Orthophosphate labeling of SH-EP1 and SHEP1 -ha4b2 cells The human clonal cell lines SH-EP1 and SH-EP1ha4b2 (stably expressing human a4b2 receptors), kindly provided by Dr. Ron Lukas (Barrow Neurological Institute, Phoenix, AZ, USA), were grown and maintained at 37 8C in 5% CO 2 . The medium for the SH-EP1 cells consisted of DMEM (high glucose) containing 5% fetal clone II, 10% horse serum, 1 mM sodium pyruvate, 8 mM L-glutamine, 100 U / ml penicillin, 100 mg / ml streptomycin, and 0.05 mg / ml amphotericin B; the medium for the SH-EP1-ha4b2 cells also included 0.5 mg / ml zeocin and 0.4 mg / ml hygromycin B for selection of the receptor. Cells were kept in continuous culture and fed every 2–3 days. SH-EP1 or SH-EP1-ha4b2 cells were grown to 85% confluence in 60 mm dishes. To label endogenous ATP stores, the plates were washed three times with 2 ml of phosphate-free DMEM containing 8 mM L-glutamine, 100 U / ml penicillin, and 100 mg / ml streptomycin, and the cells were incubated in media containing 0.0625 mCi / ml 32 P-orthophosphate for 4 h.

2.3. Membrane preparation and immunoprecipitation of a4 subunits Cells were harvested by washing the plates five times

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with 2 ml of ice-cold phosphate-buffered saline before addition of homogenization buffer [200 mM potassium phosphate, 150 mM NaCl, 10 mM EDTA, 10 mM EGTA, 10 mM b-glycerophosphate, 50 mM NaF, 1 mM NaVO 3 , 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mg / ml pepstatin A, 10 mg / ml leupeptin, and 10 U / ml aprotonin]. The plates were scraped and the cells triturated by mechanical disruption (20 strokes) using a 1-ml syringe and 26-gauge needle, followed by centrifugation at 500 g for 5 min at 4 8C. The supernatant was centrifuged at 20,800 g for 30 min at 4 8C, and the membrane pellet obtained was resuspended in homogenization buffer containing 2% Triton X-100 and solubilized for 30 min at 4 8C. Samples were centrifuged at 230,000 g for 10 min at 4 8C and the 32 P-labeled solubilized proteins were used for immunoprecipitation. The a4 subunits were immunoprecipitated from the detergent extracts by rotating overnight at 4 8C with 5 mg of mAb299 (monoclonal antibody to the rat a4 subunit that also recognizes the human a4 subunit) pre-coupled to protein-G Sepharose beads. The bead / antibody / receptor complex was washed five times with final storage buffer [50 mM 3-(N-morpholino)propanesulfonic acid (MOPS), 1 mM Na 2 EDTA, 1 mM EGTA, pH 7.2 and 0.2% Triton X-100], and resuspended in a final volume of 20 ml with 23 Laemmli buffer [13].

2.4. Autoradiography and immunoblotting The immunoprecipitated proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) on 7.5% polyacrylamide gels and transferred to PVDF membranes. The incorporation of 32 Porthophosphate was visualized by autoradiography by exposing the PVDF membranes to Kodak XAR film in the presence of intensifying screens at 280 8C for 18–24 h before immunoblotting. A rabbit polyclonal antibody generated against a recombinant protein corresponding to amino acids 342–474 mapping near the carboxy terminus of the human a4 receptor subunit was used to determine the relative quantity of a4 receptor subunit protein. PVDF membranes were blocked for 2 h at room temperature using Tris-buffered saline (TBS) containing 0.05% Tween 20 and 5.5% nonfat dry milk, and were incubated with primary antibody (4 mg / ml) in TBS / 0.05% Tween / 0.5% non-fat dry milk for 1 h at room temperature. The membranes were washed and incubated with secondary antibody (2 mg / ml; horseradish peroxidase conjugated anti-rabbit antibody) for 30 min, and the signals were visualized using enhanced chemiluminescence. Autoradiographs and immunoblots were quantitated by densitometry using a Bio-Rad Imaging Densitometer with Multi-Analyst software (Bio-Rad Laboratories, Hercules, CA, USA). Autoradiographic results were normalized to protein (immunoblots) and data were

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expressed as relative phosphorylation, i.e., the ratio of the P signal from the autoradiograph to the protein level from the immunoblot. Sigma molecular weight markers were used as a reference.

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2.5. Data analysis Grouped data are presented as the mean6S.E. Statistical significance was determined using SuperANOVA (Abacus Concepts, Berkeley, CA, USA). ANOVA (analysis of variance) was used for comparisons; 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.

3. Results The first set of experiments determined whether the a4 subunit of the human a4b2 receptor could be detected as a phosphorylated protein in situ. When SH-EP1-ha4b2 cells were incubated for 4 h with 32 P-orthophosphate, followed by immunoprecipitation of a4 subunits from a membrane preparation, results indicated the presence of a single 32 P-labeled band at approximately 80 kDa (Fig. 1, autoradiograph). The identification of this band as the a4 subunit was confirmed by Western blot analysis using a polyclonal anti-a4 receptor subunit antibody (Fig. 1, immunoblot). There were no signals on either the autoradiograph or the immunoblot from the untransfected SHEP1 cells. These results demonstrate that the a4 subunit of

Fig. 1. In situ phosphorylation of a4 subunits of the human a4b2 receptor. SH-EP1 (untransfected) and SH-EP1-ha4b2 (transfected) cells were incubated with 32 P-orthophosphate for 4 h, membranes prepared, and the a4 receptor subunit immunoprecipitated. Proteins were separated by SDS–PAGE, followed by autoradiography and immunoblot analysis. The result shown is from a single experiment that was repeated three times with similar results.

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the human a4b2 nicotinic receptor exists in the phosphorylated state in situ. To determine whether activation of PKA enhanced a4 subunit phosphorylation, SH-EP1-ha4b2 cells were incubated with 32 P-orthophosphate for 4 h, the last 15–60 min in the absence or presence of PKA activators. The a4 subunit was immunoprecipitated from a membrane preparation, and the relative phosphorylation determined. Results from these experiments are shown in Fig. 2. The a4 subunits from cells incubated in the absence of the activators exhibited a basal level of phosphorylation (in agreement with data shown in Fig. 1) which was unaffected by incubation with the dimethylsulfoxide (DMSO) vehicle (Fig. 2, top). When cells were incubated for 15 min with 10 mM forskolin, a concentration known to selectively activate the PKA signaling cascade [14], relative phosphorylation of the a4 subunit increased significantly (P,0.05) by threefold relative to the controls. Studies investigating various times of incubation (5, 10, 15, 30, 45

and 60 min) indicated that the increase was maximal following 15 min, and concentration–response studies (0.1–30 mM forskolin) indicated that 10 mM forskolin yielded a maximal effect (data not shown). Because forskolin activates PKA in an indirect manner through stimulation of adenylyl cyclase, the effects of direct activation of PKA by the cell permeable, nonhydrolyzable cAMP analogs db-cAMP and Sp-8-Br-cAMP were determined; the effects of the inactive isomer Rp-8Br-cAMP, which occupies the cAMP-binding site to prevent dissociation and activation of the kinase holoenzyme, served as an additional control. For these studies, cells were incubated during the last 60 min of 32 P-orthophosphate labeling with a concentration of the analogues that specifically activates PKA [23]. As shown in Fig. 2 (bottom), both db-cAMP and Sp-8-Br-cAMP increased a4 subunit phosphorylation significantly (P,0.05) by approximately 2–3-fold; the inactive isomer Rp-8-Br-cAMP had no effect on phosphorylation of the a4 subunit. (It should

Fig. 2. Effect of PKA activation on the phosphorylation of a4 subunits. SH-EP1-ha4b2 cells were incubated with 32 P-orthophosphate for 4 h, the last 15 (forskolin, 10 mM) or 60 (db-cAMP, 1 mM; Sp-8-Br-cAMP, 1 mM; Rp-8-Br-cAMP, 1 mM) min in the absence (basal) or presence of the PKA activators. The a4 subunit was immunoprecipitated from a membrane preparation, proteins were separated by SDS–PAGE, and samples subjected to autoradiography and immunoblot analysis. Relative phosphorylation represents the ratio of the 32 P signal determined from the autoradiograph to the protein level determined from the immunoblot. The autoradiographs and immunoblots are from a representative experiment. Values in the bar graph are the mean6S.E. from three experiments for the forskolin studies and from five experiments for the cAMP analogue experiments. * Statistically different from basal phosphorylation, P,0.05.

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be noted that differences in the scale for the relative phosphorylation signal between experiments is due to differences in the immunoblot signals of different antisera lot numbers used for different groups of experiments). To confirm that phosphorylation of the human a4 subunit is enhanced by PKA activation in situ, the ability of the selective PKA inhibitor H-89 to block the forskolininduced increase was determined (Fig. 3). For these experiments, 5 mM H-89 was used, a concentration that is 100-times the Ki of H-89 for PKA and one-sixth the Ki of H-89 for PKC or Ca 21 / calmodulin protein kinase II [19]. As shown previously, a4 subunits from cells incubated in the absence of the activators exhibited a basal level of phosphorylation that was unaffected by the DMSO vehicle, and PKA activation by forskolin (10 mM) increased the

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relative phosphorylation significantly (P,0.05) by more than twofold. When cells were incubated with forskolin in the presence of H-89 (5 mM), the forskolin-induced increase was totally prevented, and relative phosphorylation did not differ from controls. These results suggest that PKA activation, whether indirect through forskolin stimulation of adenylyl cyclase or direct via increased cAMP, promotes phosphorylation of the a4 subunit of the human a4b2 receptor in situ. Interestingly, although H-89 by itself did not produce a statistically significant effect, it decreased relative phosphorylation by 40%. The ability of H-89 to decrease phosphorylation relative to the controls suggests that a PKA-mediated process may be involved, in part, in the phosphorylation of the a4 subunit in the resting state.

Fig. 3. Effect of PKA inhibition on the phosphorylation of a4 subunits. SH-EP1-ha4b2 cells were incubated with 32 P-orthophosphate for 4 h, the last 15 min in the absence (basal) or presence of forskolin (10 mM), H-89 (5 mM), or both compounds. The a4 subunit was immunoprecipitated from a membrane preparation, proteins were separated by SDS–PAGE, and samples subjected to autoradiography and immunoblot analysis. Relative phosphorylation represents the ratio of the 32 P signal determined from the autoradiograph to the protein level determined from the immunoblot. The autoradiograph and immunoblot are from a representative experiment. Values in the bar graph are the mean6S.E. from five experiments. * Statistically different from basal phosphorylation, P,0.05.

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In addition to consensus sequence sites for PKA, the M3 / M4 cytoplasmic domain of the a4 subunit also contains predicted phosphorylation sites for PKC. To determine whether activation of PKC enhances phosphorylation of the a4 subunit, cells were incubated in the absence or presence of 200 nM PMA or PDBu during the last 30 min of labeling with 32 P-orthophosphate (Fig. 4), a time and concentration determined to produce a maximal effect (data not shown) and which has been shown to be specific for the activation of PKC in intact cells [21]. As demonstrated previously, a4 subunits from cells incubated in the absence of the activators exhibited a basal level of phosphorylation that was unaffected by the DMSO vehicle. When cells were incubated with either PMA or PDBu (Fig. 2, top), a 1.5–2-fold increase in the relative phosphorylation of the a4 subunit was observed, with the PDBu effect statistically significant (P,0.05). To ensure that the increase induced by PDBu could be attributed specifically to activation of PKC, cells were incubated with PDBu in the presence of 0.1 mM of the selective PKC inhibitor Ro-31-

8220, a concentration that has been shown to inhibit PKC without affecting PKA [4]. Ro-31-8220 completely blocked the PDBu increase, but did not, by itself, alter relative phosphorylation. These results suggest that phosphorylation of the a4 subunit of the human a4b2 receptor is enhanced upon PKC activation, but that basal phosphorylation of the a4 subunit cannot be attributed to a PKC-mediated process.

4. Discussion The present study determined whether the a4 subunit of the human a4b2 nicotinic receptor was phosphorylated in situ, and whether activation of PKA or PKC enhanced this phosphorylation. Results provide the first direct evidence that the subunit exists in a phosphorylated state in situ, and that increased phosphorylation occurs upon activation of either PKA or PKC. Furthermore, findings suggest that in the unstimulated (basal) state, subunit phosphorylation

Fig. 4. Effect of PKC activation and inhibition on the phosphorylation of a4 subunits. SH-EP1-ha4b2 cells were incubated with 32 P-orthophosphate for 4 h, the last 30 min in the absence (basal) or presence of the PKC activators PMA (200 nM) or PDBu (200 nM). For inhibition studies, cells were incubated in the absence or presence of PDBu (200 nM), Ro-31-8220 (0.1 mM), or both compounds. The a4 subunit was immunoprecipitated from a membrane preparation, proteins were separated by SDS–PAGE, and samples subjected to autoradiography and immunoblot analysis. Relative phosphorylation represents the ratio of the 32 P signal determined from the autoradiograph to the protein level determined from the immunoblot. The autoradiographs and immunoblots are from representative experiments. Values in the bar graph are the mean6S.E. from five experiments. * Statistically different from basal phosphorylation, P,0.05.

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may involve, in part, a PKA- but not a PKC-mediated process. Phosphorylation / dephosphorylation processes represent major post-translational mechanisms regulating numerous ligand-gated ion channels. During the past 20 years, many studies demonstrated that the d and g subunits of nonneuronal nicotinic receptors are phosphorylated by both PKA and PKC, and that phosphorylation / dephosphorylation plays a significant role modulating the activity of the ion channel, influencing the assembly of receptor subunits, and altering the aggregation of the receptors at synapses [15,24]. In contrast to studies documenting the importance of non-neuronal nicotinic receptor phosphorylation to receptor modulation and function, investigations of the phosphorylation / dephosphorylation of neuronal nicotinic receptors and its functional consequences have only recently begun. Results obtained from both direct and indirect studies have supported the PKA- and PKC-mediated phosphorylation of the rat a4 subunit [9,11,17,18,27,28]. The present study extends this finding to the human sequence and provides the first direct evidence that the human a4 subunit is phosphorylated in situ. Furthermore, in addition to evidence that kinase activation leads to enhanced relative phosphorylation, results also indicate that the human a4 subunit exists in a phosphorylated state in situ, an effect that may involve, in part, a PKA-mediated event. The concept that the a4b2 receptor exists in a phosphorylated state in the absence of exogenous stimulation is similar to that reported for other nicotinic receptor subunits. Indeed, both the b and d subunits of the rat muscle receptor present in myotubes have been shown to be phosphorylated under basal conditions, an action that was enhanced by the presence of forskolin [16]. In addition, the a3 neuronal nicotinic receptor subunit expressed in chicken ciliary ganglion neurons exhibits a basal level of phosphorylation [26]. The idea that a proportion of a4 neuronal nicotinic receptor subunits exists in a basal phosphorylated state suggests that two distinct populations of a4b2 receptors exist. Indeed, functional concentration–response studies have demonstrated that rat a4b2 receptors expressed in Xenopus oocytes [3] and human a4b2 receptors expressed in HEK cells [2] exist in high and low affinity states. If subunit phosphorylation alters the affinity of the receptor for agonist and modifies the functional characteristics of the channel, then it is possible that this post-translational modification is responsible for functionally distinct receptors with different affinity states reflecting phosphorylated and non-phosphorylated populations of receptors. Thus, although it is evident that the a4 subunit exists in both a phosphorylated and non-phosphorylated state, the relationship between these biochemical states and observed functional states remains to be investigated. The most compelling evidence supporting a functional consequence of a4 subunit phosphorylation comes from studies demonstrating that the PKA-mediated phosphoryla-

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tion of the a4 subunit enhances its affinity for the 14-3-3 chaperone protein, resulting in increased expression of the a4b2 receptor [12]. The 14-3-3 proteins represent a family of intracellular proteins that bind to phosphoserine-containing motifs and participate in numerous regulatory mechanisms [25]. The findings that: (1) the second serine in the sequence KARSLSV contained within the M3 / M4 cytoplasmic domain of the rat a4 subunit represents a major PKA phosphorylation site [9]; (2) phosphorylation of this site enhances its interaction with 14-3-3 leading to enhanced expression of the receptor [12]; and (3) this sequence is conserved among human, rat, and mouse, suggest that it represents a major post-translational regulatory site on the receptor. In summary, this study provides the first direct evidence that the a4 subunit of the human a4b2 nicotinic receptor is phosphorylated in situ, and that activation of PKA and PKC enhance phosphorylation. Ongoing studies are identifying the amino acid residues phosphorylated and investigating the functional significance of this post-translational modification.

Acknowledgements This research was supported by NIDA grant DA14010 (to L.W.) and the Chiles Endowment Biomedical Research Program of the Florida Department of Health BM038 (to L.W.). The opinions, findings and conclusions expressed in this publication are those of the authors and do not necessarily reflect the views of the Biomedical Research Program or the Florida Department of Health.

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