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Kittler JT, Moss SJ, editors. The Dynamic Synapse: Molecular Methods in Ionotropic Receptor Biology. Boca Raton (FL): CRC Press; 2006.

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The Dynamic Synapse: Molecular Methods in Ionotropic Receptor Biology.

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Chapter 4Phosphorylation Site-Specific Antibodies as Research Tools in Studies of Native GABAA Receptors

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4.1. INTRODUCTION

Type A γ-aminobutyric acid (GABAA) receptors represent a large and diverse family of ligand-gated CI channels that mediate the majority of fast inhibitory neurotransmission in the brain [1] by causing transient reduction in the probability of action potential firing due to plasma membrane hyperpolarization. Modifications of GABAA receptor function are believed to be critical for development and coordination of neuronal activity underlying all physiological and behavioral processes, and the vast majority of neurons in the brain express these receptors on their plasma membrane. Native, bicuculline-sensitive GABAA receptors are hetero-pentamers of subunits with multiple isoforms classified as: α (1–6), β (1–3), γ (1–3), δ, epsilon, π and θ [2–4], with a common transmembrane topology comprising a large N-terminal domain, four transmembrane domains (TMs), and a major intracellular domain between TMs 3 and 4 [5] (Figure 4.1b and Figure 4.1c). Some of the most common subunits, such as β3 [6] and γ2 [7] are essential for synaptic inhibition and organism survival. In contrast, individual isoforms of the α subunit are highly specialized to maintain function of neuronal circuits associated with specific behavioral phenotypes, including sedation, memory, anxiety, arousal and others [8,9]. GABAA receptors are also the main sites of action for a variety of compounds with potent sedative, hypnotic, anxiolytic and anticonvulsant effects, such as benzodiazepines, barbiturates, neurosteroids and anaesthetics [10,11].

FIGURE 4.1. Structural hierarchy in organization of native GABAA receptors.

FIGURE 4.1

Structural hierarchy in organization of native GABAA receptors. (a) GABAA receptors are enriched at the post-synaptic membrane opposing the pre-synaptic GABA releasing terminal where they form clusters of functional receptor molecules. (b) The prototypical (more...)

One of the key morphological features of GABAergic synapses allowing for the high efficacy of stimulus/transmission coupling is the presence of GABAA receptors clustered at the post-synaptic membrane opposite of GABA-releasing presynaptic terminals (Figure 4.1a). Targeting of GABAA receptors to these sites is facilitated, at least in part, by interactions with a variety of regulatory proteins [12], whereas their stabilization at post-synaptic sites is partially dependent on multifunctional protein gephyrin [13,14]. Receptor removal from synaptic sites, largely via clathrin-dependent endocytosis, is mediated by dynamic association with the AP2 adaptin complex [15]. Intracellular trafficking of GABAA receptors is thus critical for shaping the response of the post-synaptic neuron to released GABA and operates over the time scale of tens of minutes to hours [16]. In contrast, the acute and transient modulation of GABAA receptor function in a variety of neuronal cell types, occurring within seconds or minutes, is believed to involve a reversible modification of GABAA receptor subunits via direct phosphorylation. However, at the present time, the direct evidence for phosphorylation of native GABAA receptors is just beginning to emerge and it is facilitated by the production of phosphorylation state-specific antibodies designed to bind to specific GABAA receptor subunits.

The effects of activation of various signal transduction pathways have been shown to range from potentiation to inhibition of GABA-gated currents in different types of neurons [17]. For example, activation of protein kinase A (PKA) in cultured superior cervical ganglia neurons, spinal cord neurons, cerebellar granule cells, hippocampal pyramidal cells and olfactory bulb granule cells [18–22] has been demonstrated to correlate with a decrease in GABA-activated currents. In contrast, activation of PKA in hippocampal dentate granule neurons, cerebellar Purkinje neurons and olfactory bulb granule cells has been correlated with an enhancement of GABA-activated currents [23,24]. Similar cell type-specific differences have been observed by activation of various G-protein coupled receptors (GPCR). For example, activation of D1 receptors by specific agonists, or injections of cAMP analogues, were both found to cause a decrease in GABA-activated currents in acutely dissociated neostriatal neurons. The role of PKA in this process was confirmed by the application of specific PKA inhibitors [22]. Similar PKA-dependent inhibition of GABA-activated currents was detected in olfactory bulb granule cells in response to D1 receptor activation by dopamine. In contrast, in olfactory bulb mitral cells, dopamine was shown to enhance GABAA receptor function through the activation of D2 receptors and concomitant stimulation of Ca2+/phospholipid-dependent protein kinase (PKC) [25]. One possible explanation for the diversity of functional effects in different neuronal cell types is the functional compartmentalization of D1 and D2 receptors with different GABAA receptor subtypes. Establishing whether these differences are the direct consequence of changes in the phosphorylation state of different GABAA receptor subtypes, or involve alternative mechanisms such as activation of different signalling pathways, variations in levels of kinases and phosphatases and their anchoring molecules [26,27], or phosphorylation of other proteins associated with GABAergic synapses is important. Diverse functional effects on GABA-gated currents were also recorded in response to elevated intracellular Ca2+ levels and, again, these effects were found to depend on a neuronal cell-type studied [28,29]. Downstream of cytosolic Ca2+ increases, both Ca2+/calmodulin-dependent protein kinases (CaM kinase II) and Ca2+/calmodulin-dependent protein phosphatases (protein phosphatase 2B/calcineurin) could underlie the regulation of GABA-currents, given that both classes of signalling molecules were shown to modulate GABAA receptor phosphorylation state, at least in vitro, by phosphorylating [30] or dephosphorylating [31] several sites in β and γ subunits. CaM kinase II and PKA were independently found to play important roles in the phenomenon known as “rebound potentiation” of GABAergic currents in cerebellar Purkinje neurons, where glutamatergic stimulation leads to an increase in intracellular Ca2+ and an enhancement of GABAergic currents [23,32]. In some neuronal cell types, activation of tyrosine kinases was correlated with functional modulation of GABAA receptors. The increase in the amplitude of mIPSCs in hippocampal neurons and brain slices in response to insulin was proposed to be mediated by both tyrosine [33] and serine/threonine-specific kinases [31]. Similar enhancement of GABAergic currents was observed in cultured superior cervical ganglion (SCG) neurons and spinal cord neurons in response to c-src activation [34]. In Purkinje neurons, the activation of TrkB receptor by brain-derived neurotrophic factor BDNF was found to promote the effect of c-src tyrosine kinase activation, resulting in an increase in the amplitude of mIPSCs [35].

Notwithstanding the aforementioned evidence for the role of phosphosphorylation/dephosphorylation in the modulation GABAAR function, among the large number of GABAA receptor subunit classes, members of only two of these classes, β and γ subunits, have been demonstrated to comprise highly conserved serine, threomine and tyrosine residues localized within the intracellular loop between TMs 3 and 4 [17]. Early in vitro studies using intracellular domains of various β and γsubunits prepared as GST-fusion proteins, have indicated that these residues are in vitro substrates for a variety of kinases including: PKA, PKC, CaM kinase II, cGMP-dependent protein kinase (PKG) and the non-receptor tyrosine kinase, c-src [36]. Individual amino acid residues phosphorylated in vitro have been characterized by site directed mutagenesis. One remarkable result from these in vitro phosphorylation studies was the observation that multiple protein kinases appear equally capable of phosphorylating the same set of phosphorylation sites in the β and γ subunits of GABAA receptors (Figure 4.2).

FIGURE 4.2. In vitro phosphorylation of GABAA receptor subunits by multiple protein kinases.

FIGURE 4.2

In vitro phosphorylation of GABAA receptor subunits by multiple protein kinases. Individual phosphorylated residues within the TM 3 and 4 intracellular loops of β1, β2, β3 and γ2 subunits, and corresponding protein kinases, (more...)

Some of the phosphorylation studies with GST-fusion proteins of β and γ subunit domains have been recapitulated within the context of the full-length sequence of the receptor subunits expressed in heterologous expression systems (HEK293 cells, COS-7 cells), and correlated further with functional modulation of GABAA receptors. Phosphorylation of β1, β2 and β3 subunits by PKA on conserved serine residues (Ser409/410 present in all three β isoforms, together with Ser408 present only in the β3 subunit) was correlated with a bidirectional modulation of GABA-gated currents which was shown to depend on the type of β subunit expressed [37]. Thus, the functional outcome of β1 subunit phosphorylation on Ser409 appeared to be a decrease in GABA-gated currents, whereas β3 subunit phosphorylation on Ser408 and Ser409 led to an enhancement in GABA-gated currents. Phosphorylation of both β and γ subunits of GABAA receptors by PKC was demonstrated to cause an inhibition in GABA-evoked currents in heterologous systems [38], whereas phosphorylation of conserved tyrosine residues in the γ subunit by c-src resulted in an increase in GABA-gated currents [34].

Given the potent effects of direct phosphorylation of GABAA receptors on their functional properties in heterologous systems [37,38], studying the phosphorylation-dependent mechanisms regulating GABAA receptors in neuronal systems and their modulation by physiologically relevant neuron-specific extracellular and intracellular signals is imperative. Currently, the most advanced understanding of signalling mechanisms regulating native GABAA receptors is based on biochemical and electrophysiological evidence for a direct association of PKCβII and receptor for activated C kinase (RACK-1) with the β1 through 3 subunits. The binding of PKC to the β subunits has been localized to the sequence encompassing the major phosphorylation sites, Ser408/409(410) [39]. RACK-1 was found to bind independently of PKC to the sequence in the β subunits localized immediately upstream of the PKC binding sequence [39]. RACK-1 binding stimulates PKC activity, leading to a further increase in phosphorylation state of β subunits on Ser408/409 [17]. In addition, PKA signalling pathways have also been implicated because A-kinase (PKA) anchoring proteins (AKAPs) mediate binding of PKA to some GABAA receptor subtypes [40].

Thus, direct phosphorylation of GABAA receptors has emerged as an essential mechanism for the dynamic modulation of the channel activity and might contribute to long-term changes in neuronal function, such as those experimentally observed as long-term potentiation/depression, and viewed as cellular correlates of learning and memory [26]. The complex regulation of GABAA receptor function in situ, in correlation with the phosphorylation of GABAA receptors by multiple protein kinases, at least in vitro, raises many important questions regarding the specificity of GABAA receptor regulation by phosphorylation, such as the mechanisms of spatial or temporal integration of signalling cascades as well as selection of signals. We have begun to uncover answers to some of these questions by characterizing in detail the molecular pathway that couples the activation of neurotrophin/Trk receptors to phosphorylation and functional modulation of GABAA receptors in neurons [41]. Our experiments have demonstrated that a temporally bi-phasic mode of functional modulation of GABAergic currents by BDNF is achieved by facilitating dynamic changes in GABAA receptor phosphorylation. These include acute and transient enhancement of β3 subunit phosphorylation at sites Ser408/409, followed by a rapid dephosphorylation to levels significantly lower than the phosphorylation state seen in untreated samples. These short-term biochemical and functional changes are dependent on the activities of PKC and protein phosphatase 2A (PP2A), and are mediated by transient recruitment of these signalling proteins to GABAA receptors as judged by co-immunoprecipitation. In response to BDNF, an increase in the amount of GABAA receptor–associated PKC, RACK-1 and PP2A were detected within the first 10 minutes and was followed by dissociation of PKC and RACK-1 at later time points (30 minutes in the presence of BDNF). However, PP2A remained associated with the receptor at later time points, coinciding with a rapid dephosphorylation of Ser408/409. Importantly, the extent of PKC binding to the receptor was significantly decreased by phosphorylation of Ser408/409, whereas association of PP2A was enhanced in in vitro binding assays. Phosphorylation of sites Ser408/409 thus regulates not only channel activity but also the affinity of various signalling molecules in their direct association with GABAA receptors [41].

In summary, the experimental evidence for a direct phosphorylation of native GABAA receptors is greatly facilitated by the development of phosphorylation state-specific antibodies for specific receptor subunits (Table 4.1) [31,41]. Using these powerful research tools, we have obtained the first insights into the neuronal signalling mechanisms operating at the level of GABAA receptors, pointing to a very fine mode of regulation determined by neuronal cell-type, specific subcellular compartmentalization and possibly even the history of signalling events.

TABLE 4.1

TABLE 4.1

Characterized Phosphorylation Sites in GABAA Receptor and Subunits

4.2. DESIGN, PRODUCTION AND CHARACTERIZATION OF GABAA RECEPTOR PHOSPHO-SITE SPECIFIC ANTIBODIES

Phosphorylation state-specific antibodies provide us with a remarkably sensitive and specific immunochemical tool to detect and quantify changes in the phosphorylation state of particular amino acid residues within the full-length protein sequence in intact tissue and cell preparations. These features are of particular importance in instances where a high degree of sequence similarity between various isoforms of a protein of interest, often co-expressed within the same cell type, complicate the use of more traditional methods to study in situ protein phosphorylation, for example, by prelabeling of intracellular ATP pool with 32P-orthophosphate, or by alternative post-hoc approaches such as “back” phosphorylation. Unlike these methods, the use of phosphorylation state-specific antibodies involves, in the majority of cases, relatively simple and inexpensive immunoblotting procedures. In addition, the great advantage of phosphorylation state-specific antibodies in studies of in situ protein phosphorylation becomes apparent when these antibodies can additionally be reliably employed for use in immunocytochemical studies, providing the great benefit of being able to visualize the subcellular distribution of phosphorylated forms of a protein of interest.

Following the early attempts to raise phosphorylation-dependent polyclonal antibodies by immunization with phosphorylated forms of intact proteins [42], an alternative approach utilizing phosphorylated and unphosphorylated forms of synthetic peptides was developed in the laboratory of Professor Paul Greengard at the Rockefeller University in New York, NY [43]. This method has become a general protocol for the production of phosphorylation site-specific antibodies for proteins with characterized sites of phosphorylation and it is today commercially exploited in a large-scale production of such antibodies by commercial concerns.

4.2.1. Phosphopeptide Design and Preparation

The high quality of phosphorylation site-specific antibodies is necessitated to display their specificity in two important aspects: to recognize only the protein of interest, and to recognize either the phosphorylated or unphosphorylated form with respect to a given phospho-site within this protein. Thus, one of the most critical steps in raising a new phosphorylation site-specific antibody is to determine the length (optimally up to 10 residues) and sequence of peptides that will sufficiently resemble the amino acid sequence surrounding a particular phosphorylated residue in a protein of interest but, at the same time, allow the sufficient exposure of this site as the main part of the epitope while reducing the exposure of other phosphorylation-independent residues within the epitope. The presence of charged amino acid residues such as aspartate and glutamate, as well as arginine and lysine, are known to improve the antigenicity of synthetic peptides and are often part of consensus sequences recognized by many protein kinases. In addition, the presence of two or more phosphorylated residues within the same peptide has proven to significantly increase the antigenicity of synthetic peptides. This phenomenon became evident during the production of phosphorylation site-specific antibodies for GABAA receptor β sub-units. The two most abundant β subunit isoforms (β2 and β3) contain a highly conserved stretch of basic amino acid residues within the large intracellular loop between TMs 3 and 4. This sequence includes a well-characterized phosphorylation site, Ser410, present in the β2 subunit, or Ser409, present in the β3 subunit, with one important difference between the two isoforms — namely, the β3 subunit contains an additional phosphorylation site Ser408 within the same sequence (Figure 4.3a). The presence of two phosphorylated serine residues significantly improved the antigenicity of this peptide resulting in the successful production of an antiserum specific for phospho-β3 subunit after the first round of immunization.

FIGURE 4.3. First screen: Characterization of anti-P-β3 antiserum (UCL39) using dot-immunoblotting assay.

FIGURE 4.3

First screen: Characterization of anti-P-β3 antiserum (UCL39) using dot-immunoblotting assay. (a) High degree of similarity in amino acid sequence encompassing the phosphorylation sites Ser409 in the β1 subunit, Ser410 in the β2 (more...)

Modifications of the native amino acid sequence can be introduced at the N- or C-terminus of a synthetic peptide to enable the conjugation to the carrier protein as well as direct coupling to the resin of a column for affinity purification of the antiserum. The coupling procedure is routinely based on the presence of an N- or C-terminal cysteine residue in the synthetic peptide, providing the sulfhydryl group m-maleimidodibenzoyl for coupling via m-maleimidodibenzoyl sulfosuccinimide ester bond. Thereby, the coupling reaction will occur at one end of the peptide, allowing the other end to be fully accessible as the epitope. Phosphorylated residues should ideally be positioned closer to that end of the peptide to be sufficiently exposed to the immune response. This same feature is also important for the efficient purification of the phosphorylation site-specific pool of antibodies from the antiserum using affinity chromatography, where the coupling reaction to the resin will occur at one end of the peptide, leaving the other end fully accessible to binding of specific antibodies. The alternative phospho-peptide coupling reactions are based on the presence of a tyrosyl residue at one of the ends of the peptide, thus requiring the use of bis-diazotized benzidine [44] as a coupling reagent, or the presence of epsilon-amino group of lysine residue, involving the use of glutaraldehyde as a coupling reagent [45].

Two other important determinants of peptide antigenicity are the distance of the phosphorylated residue from the free end of the peptide (N- or C-terminal) and the nature of the amino acid at the free end of the peptide; this should ideally contain a small side-chain, such as alanine [46]. Today, numerous companies provide services for the synthesis and purification of phosphorylated peptides. We routinely use the Protein Sequencing Facility at Rockefeller University. The methodologies employed by this facility have been described in detail elsewhere [46,47], and further information could be obtained online from http://pdtc.rockefeller.edu/syn/syn1.html.

4.2.2. Preparation of Phosphopeptide Antigens

For successful production of phosphorylation site-specific antibodies, phosphorylated peptide antigens are recommended to be short in sequence as previously mentioned. Due to their small size, these peptides are required to be coupled to large carrier proteins to invoke a sufficiently strong immune response. This also prevents their degradation by tissue peptidases and prolongs their half-life within the immunized animal. Here, two routinely used protocols for coupling of phosphopeptides to the carrier proteins are described.

4.2.2.1. Phosphopeptide Coupling Protocol Based on Terminal Cysteine Residue

Cysteine-containing phosphopeptides are coupled to the carrier proteins using the heterobifunctional cross-linker sulfo-MBS (m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, Pierce). Sulfonated NHS-ester cross-linkers, supplied as sodium salts, are water soluble, non-cleavable and membrane impermeable. The maleimide group is the most selective for sulfhydryl group when the pH of the reaction is kept between 6.5 and 7.5, and a stable thioether linkage formed in this reaction cannot be cleaved under physiological conditions. The NHS group of sulfo-MBS is first coupled to free amino groups (N-terminal and epsilon-amino group of lysine) in the carrier protein and then, after excess cross-linker is removed by a desalting column, the cysteine-containing epitope-peptide is coupled via reaction with the maleimide group. The most common carrier proteins in use are: Limulus hemocyanin (LHC, Sigma), Keyhole limpet hemocyanin (KLH, Sigma), bovine thyroglobulin (Sigma) and bovine serum albumin (BSA, Sigma). The protocol for coupling [46] is briefly summarized below.

  1. Reconstitute 4 mg of the carrier protein in 2 ml of 75 mM Na-phosphate, pH 7.2/250 mM NaCl. Filter the protein solution through the 0.45 μm syringe filter (Millipore).
  2. Dissolve sulfo-MBS immediately prior to use in the same buffer to obtain 2.5 mg/ml stock solution.
  3. Add 125 μl of this solution to 1.25 ml of carrier protein solution and incubate for 60 min at room temperature.
  4. Prepare one 10-ml Speedy desalting column (Pierce) by equilibrating it with about ~70 ml of the buffer.
  5. Load carrier protein/MBS solution onto the column and elute 0.5 ml fractions with 0.5 ml aliquots of the buffer.
  6. Determine the protein content by using Bradford assay or reading the absorbance of each fraction at λ280. Pool two or three fractions with the highest protein content to make a final volume to 1.5 ml.
  7. Dissolve 2 mg of peptide in 200 μl of buffer and perform two important checks before continuing with the coupling procedure. First, check the pH of the peptide solution and adjust it to pH 7.0 if acidic (this often results from the HPLC-purification of the peptide). Second, check the presence of free sulfhydryl group using Ellman’s reagent, 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB, Pierce) and measuring the absorbance at λ412.
  8. Add the peptide solution to the solution of activated carrier protein and incubate for 2 hours at room temperature.
  9. Dilute the mixture to a final volume of 1.8 ml with H2O and prepare the following aliquots: 0.6 ml aliquot for initial immunization of two rabbits (equal to 300 μg of phosphopeptide/rabbit), and four aliquots of 0.3 ml for subsequent boosts (equal to 150 μg of phosphopeptide/rabbit). Store aliquots at 20°C.

4.2.2.2. Phosphopeptide Coupling Protocol Based on Terminal Lysine Residue

Phosphopeptides containing either a free NH2-terminal group or epsilon-NH2 group of a lysine residue can be coupled to the carrier protein using glutaraldehyde as a cross-linking reagent. The coupling efficiency could vary with the molecular weight of the peptide and weight-percent of the peptide, assuming that peptides are likely to be 75% pure by weight. Peptide-to-carrier protein ratio should optimally be 25:1. This protocol employs bovine thyroglobulin (Sigma) as a carrier protein. The protocol for coupling is briefly summarized below.

  1. Reconstitute 33 mg of thyroglobulin in 3 ml of 125 mM sodium buffer, pH 7.5, and filter through 0.45-μm syringe filter (Millipore) into a small glass vial containing a small magnetic stirring bar.
  2. Dissolve peptide in 200 μl H2O to check the solubility and determine the pH with indicator paper. If necessary, adjust the pH to 7.5.
  3. Place vial on magnetic stirrer in a cold room.
  4. Dilute 25% glutaraldehyde stock (Sigma) to 0.2%. Add 2.5 ml glutaraldehyde slowly while stirring over a 20 min period. Incubate further for total of 120 min.
  5. Dissolve 10 mg of Na-borohydride (Sigma) in 800 μl of H2O. Add 150μl of this solution to the stirred vial to quench the coupling reaction. Note that the solution will tend to foam as H2 gas is released. Stir for 30 min before dialysis.
  6. Dialyse solution against 2 l of 10 mM PBS, pH 7.5. After dialysis, prepare the following aliquots: one 1 ml aliquot for the initial immunization of two rabbits and four aliquots of the remaining solution for subsequent boosts of two rabbits.

4.2.3. Rabbit Immunization Protocol

Today, a large number of companies provide services for the immunization of rabbits and production of antisera. We routinely use services provided by Cocalico Biologicals Inc. of Philadelphia, PA (http://www.cocalicobiologicals.com). The procedure used is briefly summarized here. Prior to injection, pre-immune serum is collected from each rabbit. The immunization procedure starts with the intradermal injection of the phosphopeptide-carrier protein solution (300 μg for initial immunization/rabbit) mixed with complete Freund’s adjuvant (Difco). This is followed by two booster injections (150 μg/rabbit) during the third and fourth week of the procedure. The first bleed is collected during the fifth week. The third boost is given during the sixth week and second bleed collected during the seventh week. Serum is then tested and further boosts depend on the level of the immune response. If the response is negative after four or more boosts, continuing the immunization procedure with an alternative immunogen containing the same phosphopeptide conjugated to a different carrier protein is recommended. The titer of antibodies should be monitored regularly and the final exsanguination of the rabbit should be preceded by a final boost with the antigen.

4.2.4. Phosphorylation State-Specific Antisera Screening and Purification

Newly obtained antiserum is subjected to a series of assays in order to be fully characterized as a tool with which to monitor and quantify the phosphorylation state of a protein of interest. Here, the production and characterization of GABAA receptor anti-P-β3 antibody is described in detail. The amino acid sequence of a phosphopeptide used for immunization is depicted in the Figure 4.3a. In addition to testing for specificity of this antibody toward the phosphorylated form of the β3 subunit, we have also tested whether this antibody binds preferentially to the diphospho form of the β3 subunit, containing both P-Ser408 ands P-Ser409 residues, in comparison with single phospho forms of this subunit, containing either P-Ser408 or P-Ser409. This additional level of specificity was important to determine at this stage to ensure that the P-β3 antibody specifically binds to the phosphorylated β3 subunit, the only isoform of β subunits containing two phosphorylated residues, without cross-reacting with the β1 and β2 subunits.

4.2.4.1. First Screen: Dot-Immunoblotting of Synthetic Peptides

The initial screen of a newly obtained antiserum is routinely carried out using a dot-immunoblotting assay, which employs both dephospho and phospho forms of the peptide used as antigen. The result of this screen usually indicates the specificity of the antiserum toward the phosphorylated peptide, as well as the titre of the phospho-specific pool of antibodies within the antiserum. The dot-immunoblotting assay [46] used for the initial characterization of the GABAA receptor anti-P-β3 antiserum is described below.

The primary screen of the GABAA receptor anti-P-β3 antiserum (UCL39) was performed using increasing amounts of dephospho-, P-Ser408-, P-Ser409- and P-Ser408/409 peptides (Figure 4.3b). Peptides were dissolved in 50 mM HEPES, pH 7.6, and increasing amounts (5, 25, 50 and 100 ng) spotted directly onto the polyvinylidiene difluoride (PVDF) membrane (Immobilon P, Millipore). The binding of peptides was confirmed by probing the membranes with Ponceau S protein stain. Immunoblotting was carried out in 50 mM Tris, pH 7.5/200 mM NaCl/0.05% (v/v) Tween-20 (Sigma). To reduce the nonspecific binding, membranes were first incubated in the same buffer containing 2 mg/ml BSA for 30 min. This step was followed by a 90 min incubation with the following test bleeds (all at the dilution of 1:100): pre-immune serum, first and second test bleed. Membranes were washed four times and incubated further in the presence of an anti-rabbit secondary antibody coupled to alkaline phosphatase (Promega) for 60 min, at a dilution of 1:1000. Membranes were washed four times with TBS/T, followed by two washes with TBS and a single wash with alkaline phosphatase buffer (100 mM Tris, pH 9.5/100 mM NaCl/10 mM MgCl2). Two color substrates specific for alkaline phosphatases — nitro blue tetrazolium (NBT, 35 μl/5 ml of buffer, Promega) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP, 17.5 μl/5 ml of buffer, Promega) — were added and the purple precipitate was developed to visualize the binding of antibodies to dot-blotted peptides. The results of this assay are presented in Figure 4.3b.

This initial screen provides us with important information as to whether and how to proceed with the immunization protocol. Often the antiserum contains a mixture of Immunoglobulin G (IgG) with the reactivity toward both phospho- and dephospho-peptide, and the ratio of different IgG pools can change during the course of immunizations and boosts. However, even if the result of dot-immunoblotting assay indicates the presence of high-titer antibodies, partially or completely specific for the phosphorylated form of the peptide, secondary tests must be carried out to confirm the immunoreactivity toward the same phosphorylation sites within the full-length sequence of a protein of interest.

4.2.4.2. Second Screen: Immunoblotting of Dephospho/Phospho Forms of the Holoprotein

Secondary screening of a newly obtained antiserum requires the availability of sufficient purified amounts of the protein of interest. However, if the native holo-protein is not easily available, its recombinant form expressed either as a holoprotein or as a large fragment of the protein can be used. Most commonly, these proteins are purified from E. coli as GST-tagged or His-tagged fusion-proteins. For this screen, purified protein is in vitro phosphorylated at specific phosphorylation sites to a high stoichiometry (phospho). Control, dephosphoprotein is incubated under the same conditions but in the absence of the kinase activity (mock-phospho). Increasing amounts of both mock-phospho and phospho proteins (0.1 to 1 μg) are then directly spotted onto the PVDF membrane and dot-immunoblotting is performed. Alternately, proteins can be resolved by SDS-PAGE and transferred onto the nitrocellulose membrane (Schleicher and Schuell). Immunoblotting assay is carried out as described in the previous section.

The secondary screen carried out to confirm the specificity of GABAA receptor anti-P-β3 antiserum (UCL 39) employed GST-fusion proteins containing the TM3-TM4 intracellular loop of the β3 subunit (GST-β3il), due to the limited availability of the holo-β3 subunit expressed as a GST-fusion protein. GST-β3il protein was in vitro phosphorylated at Ser408/409 by purified PKC to a stoichiometry of 0.85 mol P/mol protein (phospho). Mock-phospho protein was incubated under the same conditions in the absence of the kinase. Increasing amounts of both mock-phospho-and phospho-GST fusion proteins (100 and 200 ng) were resolved using SDS-PAGE and transferred onto the nitrocellulose membrane. Immunoblotting assays using 1:100 dilution of UCL39 were carried out as described in the previous section. The primary antibody binding was detected using [125I]-labeled anti-rabbit secondary antibody (Amersham) and Phosphoimager scanning (Molecular Dynamics, Figure 4.4). The secondary screen of UCL39 confirmed the exclusive binding of this antiserum to the phosphorylated form of the GABAA receptor β3 subunit.

FIGURE 4.4. Second screen: Characterization of anti-P-β3 antiserum (UCL39) using in vitro phosphorylated GST-β3il fusion protein.

FIGURE 4.4

Second screen: Characterization of anti-P-β3 antiserum (UCL39) using in vitro phosphorylated GST-β3il fusion protein. GST-β3il protein was in vitro phosphorylated at Ser408/409 by purified PKC and increasing amounts (100 and 200 (more...)

For any further analysis of a newly raised phosphorylation state-specific antiserum, isolating a pool of IgGs that specifically binds to the phosphorylated epitope of the protein of interest is important. The methods routinely used to purify a phosphorylation site-specific antibody using affinity chromatography will be described in a later section.

4.2.4.3. Third Screen: Expression of wt and Phospho-Site Mutants in Heterologous Cell Line Systems

GABAA receptor β subunits exhibit a high degree of similarity in the sequence surrounding the conserved phosphorylation sites Ser408 and Ser409 (or Ser410 in the β2 subunit). The strategy used to raise a phosphorylation site-specific antibody, which would be both phospho-site specific and subunit-specific in this case, was based on the presence of two phosphorylated residues, Ser408 and Ser409, exclusively in the β3 subunit. Once the primary and secondary screens confirmed the specificity of antiserum UCL39 toward the phospho forms of the β3 subunit, the third screen was designed to test whether the affinity-purified UCL39 antiserum, referred to as anti-P-β3 antibody, specifically recognized the β3 subunit only when both Ser408/409 residues were phosphorylated. This screen employed the expression of recombinant β3 subunit containing the wt sequence including both Ser408 and Ser409 residues and mutated β3 subunits containing the following serine to alanine substitutions: S408-A, S409-A and S408/409-A. These constructs were tranfected into the COS-7 cell-line and the expression levels were tested after 48 hours. Prior to preparation of SDS lysates (2% final concentration), cell cultures expressing each of the constructs were incubated in the absence or presence of forskolin to activate endogenous PKA and stimulate phosphorylation of these sites. Protein samples were resolved by SDS-PAGE, followed by transfer onto the nitrocellulose membrane. Immunoblotting assays using 0.5 μg/ml of anti-P-β3 antibody and 1 μg/ml of β3-specific, phosphorylation state-independent antibody were carried out in parallel, as described in an earlier section. Immunoblotting with anti-P-β3 Ab revealed a specific band of about ~58 kDa present only in lysates from COS-7 cells transfected with the wt β3 subunit under control conditions, which was strongly enhanced by forskolin (Figure 4.5, lane 3 (−) (+) Forsk, respectively). The total level of transfected wt β3, as detected by β3-specific antibody, remained constant (Figure 4.5, lane 3). The anti-P-β3 immunoreactive band was absent in lysates from mock transfected controls, cells transfected with α1 and γ2 subunits only, or cells transfected with either the single-phospho-site (β3S408A or β3S409A), or di-phospho-site (β3S408A,S409A) β3 mutants (Figure 4.5, lanes 1, 2, 4, 5 and 6, respectively). The expression levels of these mutants were similar to those in wt β3 transfected cells (Figure 4.5). Some weak nonspecific bands of higher molecular weight were detected with the anti-P-β3 Ab in COS-7 cell lysates, but these bands did not represent GABAA receptor β3-specific immunoreactivity as they were also detected in extracts from mock-transfected cells. These results have provided us with the key evidence for a special feature of the anti-P-β3 antibody to recognize phosphorylated β3 subunit, but only when specifically phosphorylated at both Ser408 and Ser409 residues.

FIGURE 4.5. Third screen: Characterization of anti-P-β3 antibody using the recombinant wt β3 subunit and phosphorylation site Ser408/409 β3 mutants in COS-7 cells.

FIGURE 4.5

Third screen: Characterization of anti-P-β3 antibody using the recombinant wt β3 subunit and phosphorylation site Ser408/409 β3 mutants in COS-7 cells. Recombinant wt β3 subunit (lane 3) and its phosphorylation site mutants: (more...)

The foregoing type of analysis has proven to be a very important step during the characterization of a newly raised phosphorylation state-specific antibody because it can provide clear evidence for the specificity of the antibody for particular phosphorylated residues within the full-length sequence of the protein of interest. However, the successful application of this type of analysis is dependent on the level of endogenous phosphorylation of a protein of interest in transfected cells, which can often be too low to detect under basal conditions. This problem could be circumvented by either activating the endogenous kinase activity responsible for phosphorylation (if known) using specific activators, or by co-transfecting a constitutively active form of the kinase together with the protein of interest. Alternatively, cells can be treated with a broad spectrum phosphatase inhibitor, such as high doses of okadaic acid, resulting in an enhancement of the endogenous phosphorylation state of a protein of interest, thereby increasing the probability of detection by a phosphorylation state-specific antibody.

Further important information that can be obtained from this type of assay is the determination as to whether the newly raised phospho-specific antibody can be reliably employed as a quantitative tool for analyzing the in situ phosphorylation state of the protein of interest. In this context, noting here that a significant number of attempts to raise new phosphorylation state-specific antibodies fail to pass this important step of characterization is important.

4.2.4.4. Fourth Screen: Dephospho/Phospho Peptide Block

Our final goal is to obtain a phosphorylation state-specific antibody that could be utilized as a sensitive, specific and quantitative tool with which we can detect the endogenous phosphorylation level of a protein of interest in tissue or cells where this protein normally operates. Therefore, the final checkpoint for a newly raised phospho-antibody is to determine if this antibody specifically detects the phosphorylated native form of the protein of interest. This analysis involves immunoblotting with phosphorylation state-specific antibody in the absence or presence of a dephospho- or phospho-form of the peptide used as antigen. Here, this final step is described with respect to anti-P-β3 antibody characterization that was carried out by immunoblotting of protein extracts from cultured cerebrocortical neurons, incubated either under control condition or in the presence of BDNF (100 ng/ml).

Anti-P-β3 antibody was incubated in the absence or presence of dephospho Ser408/409-peptide antigen or phospho Ser408/409-peptide antigen (both in 1:1000 antibody-to-peptide ratio) prior to the immunoblotting step. Using this approach, we detected a single band of about ~58 kDa in SDS extracts from cortical neurons identical in molecular mass to the recombinant β3 subunit (Figure 4.6, left panel) when antibody was incubated in the absence of antigen peptides. In the presence of a synthetic peptide phosphorylated at both Ser408/409, detection of the β3 subunit by anti-P-β3 Ab was prevented (Figure 4.6, right panel). In contrast, the detection of β3 subunit by anti-P-β3 Ab was unaffected by the presence of dephospho-Ser408/409 peptide (Figure 4.6, middle panel). Note that the BDNF-dependent increase in β3 subunit phosphorylation detected with the anti-P-β3 antibody (approximately four-fold, Figure 4.6) appeared quantitatively similar to the increase in phosphorylation detected by immunoprecipitation of β3 from lysates of [32P]-labeled neurons using a β3-specific antibody [41]. Together, these results from independent measures demonstrated that anti-P-β3 Ab specifically recognizes the native GABAAR-β3 subunit only when phosphorylated on both Ser408/409 residues.

FIGURE 4.6. Fourth screen: Characterization of anti-P-β3 antibody using dephospho/phospho-peptide block approach.

FIGURE 4.6

Fourth screen: Characterization of anti-P-β3 antibody using dephospho/phospho-peptide block approach. Cultured cerebrocortical neurons were incubated under control conditions (con), or in the presence of brain-derived neurotrophic factor (BDNF, (more...)

4.2.5. Affinity-Purification of Phosphorylation Statespecific Antibodies

Further purification of a subpopulation of phosphorylation state-specific IgGs from the whole serum is often required due to the presence of nonspecific IgGs recognizing other proteins in cell extracts and potentially affecting precise quantification of the phosphorylated-form of a protein of interest. The second common reason for further purification of phospho-specific IgGs is the presence of low titer IgGs cross-reacting with other epitopes present in the antigen peptide, giving rise to a low-level binding to dephospho form of the protein of interest in cell extracts.

The isolation of a phospho-specific pool of IgGs from the whole serum commonly includes two steps of purification, the first step being purification of total IgGs from the antiserum followed by the second step of purification that is based on the affinity chromatography using phosphopeptide or dephosphopeptide columns [46].

4.2.5.1. Purification of Total IgGs from the Phospho-Specific Antiserum

Purification of total pool of IgGs from the antiserum is performed as the first step in order to prevent rapid dephosphorylation as well as degradation of the phospho-peptide antigen coupled to the affinity column by high activities of phosphatases and proteases present in the whole serum. This purification step is carried out using Protein A-Sepharose beads (Protein A Sepharose CL-4B, Pharmacia), which bind to the Fc region of IgGs. The binding capacity of this matrix is high (about 20 mg IgG/ml of gel). The required amount of beads is determined by the volume of gel required for column preparation, with the assumption that 1 g of dry beads will produce about 4 to 5 ml of the final gel volume. Sepharose beads are supplied as freeze-dried gel in the presence of additives. Beads are therefore reconstituted in water and thoroughly washed to remove these additives. The column prepared with the washed bead slurry is equilibrated with the binding buffer (TBS/T: 50 mM Tris, pH 7.5/500 mM NaCl/0.05% Tween-20) in a ratio of 75% settled gel to 25% buffer. The protocol for purification is described below:

  1. An aliquot of serum (10 ml) is thawed and filtered through a 0.45-μm filter.
  2. Serum is diluted by the binding buffer to 20 ml and the following protease inhibitors are added to prevent proteolysis of antiserum: benzamidine, 25 mM; ethylene diamine tetraacetic acid (EDTA), 1 mM; ethylene glycol-bis-(beta-aminoethyl ether)-N,N- tetraacetic acid (EGTA), 1 mM; phenyl-methilsulfonyl fluoride (PMSF), 100 μM; leupeptin and antipain, 20 μg/ml; pepstatin and chymostatin, 2 μg/ml.
  3. Small aliquots of serum are loaded into the column. To increase the level of IgG binding to the column, flow-through fraction is collected and the loading is repeated.
  4. The column is washed with the following buffers (50 ml):
    50 mM Tris, pH 7.5/500 mM NaCl/0.05% Tween-20
    50 mM Na-borate, pH 8.5/500 mM NaCl/0.05% Tween-20
    50 mM Na-acetate, pH 5.5/500 mM NaCl/0.05% Tween-20
    50 mM Tris, pH 7.5/500 mM NaCl
  5. Antibodies are eluted with 100 mM glycine, pH 2.5, and 1 ml fractions are collected and neutralized with 50 μl of 1 M Tris, pH 8.0.
  6. The presence of IgGs is determined by measuring absorbance at 280 nm and fractions are pooled. The amount of purified IgGs is calculated knowing that the absorbance of 1 mg/ml of IgGs (A280) = 1.4.
  7. The column is washed and stored in the binding buffer containing 0.02% NaN2.

4.2.5.2. Purification of Phospho-Specific Antibodies Using Affinity Chromatography with Peptide Columns

The second purification step is designed to allow purification of large quantities of a subpopulation of total IgGs that show specificity toward the phosphorylated form of the protein of interest. Columns for affinity purification can be prepared using the phosphorylated protein if sufficient amount of this protein is available. However, for the long-term use of an affinity column, use of phospho- or dephosphopeptide antigen for the column preparation is recommended. Also recommended is the preparation of both phospho- and dephosphopeptide columns for affinity purification of each newly produced antiserum because the IgG pool isolated using the phosphopeptide column will likely contain a small fraction of nonspecific IgGs that bind to phosphorylation-independent epitopes of the protein of interest.

Preparation of phospho/dephospho-peptide affinity columns utilizes SulfoLink coupling gel (Pierce) for coupling of N- or C-terminal cysteine-containing peptides or activated CH Sepharose 4B (Pharmacia) for coupling of peptides via N-terminal amino group. SulfoLink coupling gel is a support matrix formed by 6% cross-linked agarose with an iodoacetyl group immobilized at the end of a 12-atom spacer. The coupling procedure [46] is briefly summarized below:

  1. Resin (about 5 ml) is washed thoroughly with about 100 ml of 50 mM Tris, pH 8.5/5 mM EDTA on a cintered glass filter.
  2. The peptide (5 to 10 mg) is dissolved in 2 ml of the same buffer and its pH and the presence of free sulfhydryl groups checked as described in an earlier section.
  3. The resin is transferred into a 15-ml plastic tube and allowed to settle.
  4. Excess washing buffer is removed and the peptide solution is added to the resin.
  5. The mixture is incubated for 1 hour at room temperature with gentle rotation.
  6. The resin is washed with about ~100 ml of the coupling buffer.
  7. The resin is incubated with 50 mM cysteine in the coupling buffer for 60 min with rotation to block remaining reactive groups.
  8. The resin is poured into a Flex column and washed with 200 ml of 1 M NaCl.
  9. Column is stored at 4°C in 50 mM Tris, pH 7.5/500 mM NaCl/0.05% Tween-20, 0.02% NaN3.

The alternative method for preparation of phosphopeptide and dephosphopeptide affinity columns is based on use of activated CH Sepharose 4B (Pharmacia). This type of resin is significantly more stable than SulfoLink and allows multiple purifications to be carried out over a long period of time. Activated CH Sepharose 4B is pre-activated gel for covalent immobilization of proteins or peptides containing primary amino groups. The matrix is formed by 4% agarose with the active group immobilized on a six-aminohexanoic acid spacer. The active group N-hydroxysuccinimide is formed by esterification of the carboxyl group of CH Sepharose 4B. The binding capacity of this matrix is high (about 6 to 16 mM of peptide/ml drained gel). The required amount of beads is determined by the volume of gel required for column preparation, with the assumption that 1 g of dry beads will produce about 4 to 5 ml of the final gel volume. The resin is supplied as freeze-dried powder in the presence of additives that must be removed before the coupling reaction. The protocol for peptide coupling is described here:

  1. Weigh out the required amount of freeze-dried powder and resuspend it in 1 mM HCl.
  2. Use about 200 ml of 1 mM HCl to remove the additive by washing the gel on a cintered glass filter.
  3. Dissolve peptide (5 to 10 mg) in coupling buffer, 0.1 M NaHCO3, pH 8.0/0.5M NaCl and check the pH of peptide solution as described in an earlier section.
  4. Add the peptide solution to the gel to form a final mix with a gel:buffer ratio of 1:2, which is suitable for coupling.
  5. Rotate the mixture for 1 to 2 hours at room temperature or 4 hours at 4°C (the use of magnetic stirrer is not recommended).
  6. Remove the excess ligand with at least 5 gel volumes of coupling buffer.
  7. Block remaining active groups with 0.1 M Tris-HCl buffer, pH 8.0, or 1 M ethanolamine, pH 8.0, for at least 1 hour.
  8. Wash the gel with at least three cycles of alterating pH, each cycle including a wash with five times the gel volume of 0.1 M acetate buffer, pH 4.0/0.5 M NaCl and a wash with five times the gel volume of 0.1 M Tris-HCl, pH 8.0/0.5 M NaCl.
  9. Wash the gel with 50 mM Tris, pH 7.5/0.5 mM NaCl/0.05% Tween-20.
  10. Pour the resin into a Flex column and store at 4°C.

The affinity purification of the phospho-specific IgGs from the antiserum is essentially carried out as described earlier. The eluted pool of IgGs should be dialyzed against PBS or 10 mM MOPS, pH 7.5/150 mM NaCl and concentrated by centrifugation using Centriprep concentrators (Amicon) to an optimal concentration of 1 mg/ml.

To isolate the sufficient quantity and quality of the phospho-specific IgGs by affinity purification, each of these protocols will likely include additional steps of purification determined by the titer as well as the specificity of the antiserum. To optimize the purification procedure adequately, all the fractions obtained during the affinity purification should be stored and tested as described in earler sections.

4.3. CONCLUDING REMARKS

In recent years, the use of phosphorylation site-specific antibodies as tools for quantitative in situ detection of specific phosphorylation sites in numerous proteins have become the method of preference for many investigators. Although a number of phosphorylation site-specific antibodies are now commercially available, the need to develop new antibodies is expected to continue to rise. This trend is in line with the rapid development of proteomic techniques and our increased understanding of the principles of signal transduction pathways and their integration. The techniques currently used for the production and characterization of phosphorylation site-specific antibodies are described in this chapter with the aim to form a working basis for the future development of new approaches and applications for these sophisticated research tools.

4.4 ACKNOWLEDGMENTS

I would like to thank Dr. Talvinder S. Sihra for critical reading of this manuscript and helpful discussions.

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