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Keywords:

  • deletion mutant;
  • G protein-coupled receptor kinase 2;
  • glutamate;
  • internalization;
  • metabotropic glutamate receptor 1a;
  • protein kinase C

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Construction of mGlu1a deletion mutants
  6. Cell culture and transfection
  7. Internalization and immunofluorescence microscopy of mGlu1a receptor constructs
  8. Inositol phosphate (IP) determination
  9. Experimental design and statistics
  10. Results
  11. Discussion
  12. Acknowledgement
  13. References

To investigate the role of the intracellular C-terminal tail of the rat metabotropic glutamate receptor 1a (mGlu1a) in receptor regulation, we constructed three C-terminal tail deletion mutants (Arg847stop, DM-I; Arg868stop, DM-II; Val893stop, DM-III). Quantification of glutamate-induced internalization provided by ELISA indicated that DM-III, like the wild-type mGlu1a, underwent rapid internalization whilst internalization of DM-I and DM-II was impaired. The selective inhibitor of protein kinase C (PKC), GF109203X, which significantly reduced glutamate-induced mGlu1a internalization, had no effect on the internalization of DM-I, DM-II, or DM-III. In addition activation by carbachol of endogenously expressed M1 muscarinic acetylcholine receptors, which induces PKC- and Ca2+-calmodulin-dependent protein kinase II-dependent internalization of mGlu1a, produced negligible internalization of the deletion mutants. Co-expression of a dominant negative mutant form of G protein-coupled receptor kinase 2 (DNM-GRK2; Lys220Arg) significantly attenuated glutamate-induced internalization of mGlu1a and DM-III, whilst internalization of DM-I and DM-II was not significantly affected. The glutamate-induced internalization of mGlu1a and DM-III, but not of DM-I or DM-II, was inhibited by expression of DNM-arrestin [arrestin-2(319–418)]. In addition glutamate-induced rapid translocation of arrestin-2-Green Fluorescent Protein (arr-2-GFP) from cytosol to membrane was only observed in cells expressing mGlu1a or DM-III. Functionally, in cells expressing mGlu1a, glutamate-stimulated inositol phosphate accumulation was increased in the presence of PKC inhibition, but so too was that in cells expressing DM-II and DM-III. Together these results indicate that different PKC mechanisms regulate the desensitization and internalization of mGlu1a. Furthermore, PKC regulation of mGlu1a internalization requires the distal C terminus of the receptor (Ser894–Leu1199), whilst in contrast glutamate-stimulated GRK- and arrestin-dependent regulation of this receptor depends on a region of 25 amino acids (Ser869–Val893) in the proximal C-terminal tail.

Abbreviations used
arr-2

arrestin-2

arrestin-2-GFP

arrestin-2-green fluorescent protein

arrestin-3-GFP

arrestin-3-green fluorescent protein

BSA

bovine serum albumin

CaMKII

Ca2+-calmodulin-dependent protein kinase II

CPCCOEt

7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester

DM-I

deletion mutant I

DM-II

deletion mutant II

DM-III

deletion mutant III

DMEM

Dulbecco's modified Eagle's medium

DNM-arr

dominant negative mutant arrestin-2

DNM-dyn

dominant negative mutant dynamin

DNM-GRK2

dominant negative mutant G protein-coupled receptor kinase 2

GPCR

G protein-coupled receptor

GRK

G protein-coupled receptor kinase

HA

haemagglutinin

HEK293

human embryonic kidney 293 cells

IP

inositol phosphate

mGlu1a

metabotropic glutamate receptor 1a

mGlu1a-GFP

metabotropic glutamate receptor 1a-green fluorescent protein

mGlu1b

metabotropic glutamate receptor 1b

PBS

phosphate-buffered saline

PKC

protein kinase C

TBS

Tris-buffered saline

Glutamate is now recognized as the major excitatory neurotransmitter in the mammalian central nervous system. In addition to its involvement in learning and memory, glutamate also has the potential to act as a potent endogenous neurotoxic agent that may play a critical role in the development or progression of diverse neurological disorders. Metabotropic glutamate receptors (mGlu receptors) are G protein-coupled receptors (GPCRs), which play a critical role in glutamate-mediated neurotransmission and synaptic plasticity events (reviewed in Schoepp et al. 1999; Hermans and Challis 2001). On the basis of their pharmacology, sequence homology and signal transduction mechanisms, metabotropic glutamate receptors have been classified into three groups, with the group I mGlu receptors, mGlu1 and mGlu5, being coupled to Gq/11 and phospholipase C. Five splice variants of mGlu1 have been described thus far, all of which differ in the length of their C-terminal tail (Pin et al. 1992; Pin and Duvoisin 1995). Members of the mGlu family bear little sequence or structural homology to other GPCRs (except Ca2+-sensing and GABAB receptors) apart from the retention of a seven transmembrane topology characteristic of GPCRs.

A widely observed feature of the GPCR superfamily is the attenuation of the receptor-stimulated signal output upon sustained or recurrent agonist stimulation, a process known as desensitization (Krupnick and Benovic 1998; Ferguson 2001). Mechanisms underlying desensitization are complex and can involve phosphorylation of the receptor, uncoupling from G proteins, internalization and ultimately intracellular down-regulation. Phosphorylation and subsequent desensitization of GPCRs can occur by two distinct mechanisms. Independent of the activation status of the GPCR, second messenger-regulated protein kinases [e.g. protein kinase C (PKC)] can phosphorylate GPCRs. In contrast, the family of G protein-coupled receptor kinases (GRKs) phosphorylate only agonist-activated GPCRs, this latter process often being referred to as homologous desensitization. Receptor phosphorylation can in turn increase the affinity of the receptor for arrestins. Arrestin/receptor binding will not only occlude receptor–G protein coupling but in many cases arrestins act as adaptors for receptor sequestration via their interaction with components of the endocytic machinery, such as AP-2 (adaptor protein 2) and clathrin, both of which are major components of clathrin-coated pits (Goodman et al. 1996; Krupnick et al. 1997a; Laporte et al. 1999). From early endosomes, receptors may then either be dephosphorylated and returned to the cell surface for another round of activation or, alternatively, enter an intracellular degradative pathway (Ferguson 2001).

A number of studies have examined the desensitization and internalization of group I mGlu receptors. The desensitization of mGlu1a, which has a long intracellular C terminus, appears to be both GRK- and PKC-dependent (Dale et al. 2000; Sallese et al. 2000; Mundell et al. 2002). Group I mGlu receptors are also known to internalize upon agonist addition (Doherty et al. 1999; Sallese et al. 2000; Mundell et al. 2002), and in recent studies we have demonstrated the arrestin- and dynamin-dependent internalization of mGlu1a, mGlu1b and mGlu1c in response to glutamate (Mundell et al. 2001, 2002). In addition to homologous desensitization, mGlu1a function can be regulated by glutamate-independent heterologous mechanisms. PKC and Ca2+ calmodulin-dependent kinase II (CaMKII) can both regulate the desensitization and internalization of mGlu1 receptors (Ciruela and McIlhinney 1997; Ciruela et al. 1999; Mundell et al. 2002). Regulation of mGlu1 splice variants by these second messenger kinases was subsequently found to confer differential arrestin dependence, with mGlu1a and mGlu1c undergoing arrestin-dependent internalization, whereas that of mGluR1b was largely arrestin-independent (Mundell et al. 2002). Thus there appear to be subtle differences in the regulation of mGlu1 splice variants that may underlie important functional roles.

In the present study, we have attempted to determine which regions of the mGlu1a receptor, and specifically its very long C terminus, mediate homologous or heterologous regulation of this receptor. To perform these studies we constructed three deletion mutants of the mGlu1a C-terminal tail (Fig. 1). We show that different regions of the C terminus of mGlu1a are critical for glutamate-induced GRK-dependent versus PKC-dependent receptor internalization. Furthermore, we show that different PKC pathways mediate desensitization and internalization of mGlu1a.

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Figure 1. Comparison of the amino acid sequences of the proposed intracellular C termini of mGlu1a, mGlu1b and the three deletion mutants; Arg847stop, DM-I; Arg868stop, DM-II; Val893stop, DM-III. All serine and threonine residues are in bold and underlined, whilst the splice site is denoted by//. Note that DM-I and DM-II are also deletion mutants of mGlu1b.

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Materials

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Construction of mGlu1a deletion mutants
  6. Cell culture and transfection
  7. Internalization and immunofluorescence microscopy of mGlu1a receptor constructs
  8. Inositol phosphate (IP) determination
  9. Experimental design and statistics
  10. Results
  11. Discussion
  12. Acknowledgement
  13. References

Dulbecco's modified Eagle's medium (DMEM) or DMEM without l-glutamine, fetal bovine serum and Lipofectamine 2000 transfection reagent were obtained from Life Technologies (Paisley, UK). Expand High Fidelity DNA polymerase, rhodamine-conjugated mouse monoclonal anti-haemagglutinin (HA) antibody (12CA5) and mouse monoclonal anti-HA antibody (3F10) were from Roche (Lewes, UK), the Rneasy RNA isolation kit was from Qiagen (Crawley, UK), murine Moloney leukaemia virus reverse transcriptase was from Promega (Southampton, UK) and pcDNA3 was from Invitrogen (Paisley, UK). Anti-HA-monoclonal antibody (HA-11), goat anti-mouse fluorescein-conjugated secondary antibody and rhodamine-conjugated transferrin were all purchased from Molecular Probes (Eugene, OR, USA). Arrestin polyclonal antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA). The myo-[3H]inositol was purchased from Amersham (Little Chalfont, UK), 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPCCOEt) was from Tocris Cookson (Bristol, UK) whilst all other reagents were from Sigma (Poole, UK).

Construction of mGlu1a deletion mutants

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Construction of mGlu1a deletion mutants
  6. Cell culture and transfection
  7. Internalization and immunofluorescence microscopy of mGlu1a receptor constructs
  8. Inositol phosphate (IP) determination
  9. Experimental design and statistics
  10. Results
  11. Discussion
  12. Acknowledgement
  13. References

An extended HA-epitope (TRMYPYDVPDYA) was introduced into the N terminus of the mGlu1a cDNA between amino acids 57 and 58 and subcloned into pcDNA3 as previously described (Mundell et al. 2001). Three C terminal tail truncation mutants of the HA-tagged mGlu1a were engineered using standard PCR techniques by introducing a stop codon following Arg847 (DM-I), Arg868 (DM-II) and Val893 (DM-III). The PCR products and pcDNA3 were digested with BamHI/NotI, and following purification, ligation was undertaken with T4 ligase overnight at 4°C. Subsequent products were transformed into DH5α cells, ampicillin-resistant colonies were amplified and the correct sequence was confirmed by sequencing.

Cell culture and transfection

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Construction of mGlu1a deletion mutants
  6. Cell culture and transfection
  7. Internalization and immunofluorescence microscopy of mGlu1a receptor constructs
  8. Inositol phosphate (IP) determination
  9. Experimental design and statistics
  10. Results
  11. Discussion
  12. Acknowledgement
  13. References

Human embryonic kidney 293 (HEK293) cells were maintained in DMEM supplemented with 10% fetal bovine serum, 100 units/mL penicillin G, and 100 μg/mL streptomycin sulfate at 37°C in a humidified atmosphere of 95% air, 5% CO2. For transient transfections, HEK293 cells were grown in 60- or 100-mm dishes to 80–90% confluence and transfected with 0.5–10 μg DNA using Lipofectamine 2000 following the manufacturer's instructions. Cells were incubated with the DNA/Lipofectamine mixture for 24 h, the media was replaced, and the cells were analysed 48 h after transfection.

Internalization and immunofluorescence microscopy of mGlu1a receptor constructs

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Construction of mGlu1a deletion mutants
  6. Cell culture and transfection
  7. Internalization and immunofluorescence microscopy of mGlu1a receptor constructs
  8. Inositol phosphate (IP) determination
  9. Experimental design and statistics
  10. Results
  11. Discussion
  12. Acknowledgement
  13. References

The mGlu1a, DM-I, DM-II and DM-III cell surface loss was assessed by ELISA as described previously (Mundell et al. 2000). Briefly, cells plated at a density of around 6 × 105 cells per 100-mm dish were transiently transfected with pcDNA3 containing mGlu1a, DM-I, DM-II, or DM-III (5 μg), in some cases with wild-type arrestin-2 (5 μg), dominant negative mutant arrestin-2 (DNM-arr, 5 μg), dominant negative mutant dynamin (DNM-dyn, 5 μg), wild-type GRK2 (5 μg), or dominant negative mutant GRK2 (DNM-GRK2, 5 μg). Twenty-four hours post-transfection, cells were split into 24-well tissue culture dishes coated with 0.1 mg/mL poly-l-lysine. Twenty-four hours later, cells were incubated with DMEM (without l-glutamine), to which glutamate (10 μm) or carbachol (1 mm) were added for up to 60 min at 37°C. In some experiments GF109203X (2 μm) was added 15 min before and then during glutamate addition. Reactions were stopped by removing the medium and fixing the cells with 3.7% formaldehyde in Tris-buffered saline (TBS; 20 mm Tris–HCl, pH 7.5, 150 mm NaCl, and 20 mm CaCl2) for 5 min at room temperature. Cells were washed three times with TBS, incubated for 45 min with TBS containing 1% bovine serum albumin (BSA), and then incubated with a primary antibody (anti-HA monoclonal HA-11, 1 : 1000 dilution in TBS/BSA) for 1 h at room temperature. Cells were washed three times with TBS, reblocked with TBS/BSA for 15 min at room temperature, and then incubated with secondary antibody (goat anti-mouse antibody conjugated with alkaline phosphatase, 1 : 1000 dilution in TBS/BSA) for 1 h at room temperature. Cells were then washed three times with TBS, and a colorimetric alkaline phosphatase substrate was added. When adequate colour change was achieved, 100 μL of sample was added to 100 μL of 0.4 m NaOH to terminate the reaction, and the samples were read at 405 nm using a microplate reader. Throughout, internalization of mGlu1a was compared against surface receptor expression at time zero. Results are expressed as either percentage surface receptor or percentage loss of surface receptor, with the background signal from pcDNA3-transfected controls subtracted from all receptor-transfected values.

Cellular distribution of mGlu1a, DM-I, DM-II and DM-III transiently transfected into HEK293 cells, was assessed by immunofluorescence microscopy (Mundell et al. 2000). Briefly, HEK293 cells, grown on poly-l-lysine-coated coverslips in six-well plates, were transiently transfected with pcDNA3 containing mGlu1a, DM-I, DM-II, or DM-III (5 μg). Forty-eight hours post-transfection, receptor distribution was assessed using a primary anti-HA-monoclonal antibody (HA-11; 1 : 200) and a goat anti-mouse fluorescein-conjugated secondary antibody (1 : 200). Coverslips were mounted using Slow-Fade mounting medium and examined by microscopy on an upright Leica TCS-NT confocal laser scanning microscope attached to a Leica DM IRBE epifluorescence microscope with phase-contrast and a Plan-Apo 40 × 1.40 NA oil immersion objective.

Arrestin-2-Green Fluorescent Protein (arrestin-2-GFP) or arrestin-3-Green Fluorescent Protein (arrestin-3-GFP) redistribution was assessed in HEK293 cells as previously described (Mundell et al. 2000). Briefly, cells were transfected as described above with 5 μg of pcDNA3 containing mGlu1a, DM-I, DM-II, or DM-III, and 0.5 μg of arrestin-2-GFP or arrestin-3-GFP and grown on poly-l-lysine-coated coverslips. To assess mGlu1a distribution, cells were incubated for 30 min at 4°C with rhodamine-conjugated mouse monoclonal anti-HA antibody (12CA5; 1 : 100). Cells were then washed three times with phosphate-buffered saline (PBS) prior to imaging and coverslips were mounted in a heated imaging chamber through which media and drugs could be added. Cells were examined by microscopy on an inverted Leica TCS-NT confocal laser scanning microscope attached to a Leica DM IRBE epifluorescence microscope with phase-contrast and a Plan-Apo 40 × 1.40 NA oil immersion objective. All images were collected on Leica TCS-NT software for two- and three-dimensional image analysis and processed on Adobe Photoshop 5.5.

Inositol phosphate (IP) determination

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Construction of mGlu1a deletion mutants
  6. Cell culture and transfection
  7. Internalization and immunofluorescence microscopy of mGlu1a receptor constructs
  8. Inositol phosphate (IP) determination
  9. Experimental design and statistics
  10. Results
  11. Discussion
  12. Acknowledgement
  13. References

This was undertaken as previously described (Mundell and Benovic, 2000). Briefly, cells plated at a density of around 6 × 105 cells in each 60-mm dish were transiently transfected with 5 μg mGlu1a, DM-I, DM-II, or DM-III, each ± arrestin-2 (5 μg). Twenty-four hours post-transfection, cells were split into 24-well tissue culture dishes coated with 0.1 mg/mL poly-l-lysine. The following day cells were labelled for 18–24 h with myo-[3H]inositol (4 μCi/mL of culture medium) in DMEM (high glucose, without inositol). After labelling, cells were washed once in PBS and incubated in pre-warmed DMEM (without l-glutamine) containing 20 mm LiCl for 10 min at 37°C. Cells were then stimulated with glutamate (10 μm) for 0–30 min. Reactions were terminated by removing the stimulation media and adding 0.8 mL of 0.4 m perchloric acid. Samples were harvested in 1.5 mL Eppendorf tubes to which 0.4 mL of 0.72 m KOH, 0.6 m KHCO3 were added. Tubes were vortexed and centrifuged for 5 min at 20 000 g in a microcentrifuge. IP were separated on Dowex AG 1-X8 columns exactly as described previously (Mundell and Benovic, 2000). Total labelled IP were determined by liquid scintillation counting.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Construction of mGlu1a deletion mutants
  6. Cell culture and transfection
  7. Internalization and immunofluorescence microscopy of mGlu1a receptor constructs
  8. Inositol phosphate (IP) determination
  9. Experimental design and statistics
  10. Results
  11. Discussion
  12. Acknowledgement
  13. References

To determine the role of the C-terminal tail in the agonist-induced regulation of mGlu1a, we constructed three C terminus deletion mutants (Val893stop, DM-III, with the final 306 amino acids deleted; Arg868stop, DM-II, with the final 331 amino acids deleted; and Arg847stop, DM-I, with the final 352 amino acids deleted; note that DM-II and DM-I are also in fact deletion mutants of mGlu1b, see Fig. 1). The relative cell surface expression of these constructs, as assessed by ELISA, was not significantly different (arbitrary absorbance units/mg cell protein: mGlu1a, 1.00 ± 0.11; mGlu1b, 0.79 ± 0.12; DM-I, 0.83 ± 0.11; DM-II, 0.80 ± 0.10; DM-III, 0.86 ± 0.10; the data are mean ± SE from 12 independent experiments). The kinetics and extent of internalization of each deletion mutant was assessed next. Quantification of glutamate-induced internalization provided by ELISA (Fig. 2a) and confirmed by immunofluorescent microscopy (Fig. 2b), indicated that DM-III underwent rapid internalization whilst internalization of both DM-I and DM-II was somewhat impaired as compared with the full-length mGlu1a (surface receptor loss with 10 μm glutamate for 30 min: mGlu1a, 32 ± 5%; DM-I, 20 ± 2%; DM-II, 20 ± 5%; DM-III, 40 ± 3%). Although it is possible that endogenous glutamate in the medium could trigger internalization of mGlu1 constructs, this was shown not to be the case because inclusion of the mGlu1a antagonist CPCCOEt in the incubation medium did not affect the cell surface expression of mGlu1a (cell surface expression of mGlu1a following 30 min pretreatment with 100 μm CPCCOEt was 98.0 ± 3.3% of untreated cells, mean ± SΕ, n = 9). Considerable evidence has accumulated that PKC activation is important for mGlu1 regulation (Ciruela and McIlhinney 1997; Ciruela et al. 1999; Francesconi and Duvoisin, 2000; Mundell et al. 2002), and so we investigated the role of PKC in the glutamate-induced internalization of the deletion mutants. In agreement with our previous studies (Mundell et al. 2002), treatment with the selective inhibitor of PKC, GF109203X significantly reduced the glutamate-induced internalization of mGlu1a but not mGlu1b (Fig. 3a). Furthermore, GF109203X treatment had no effect on the glutamate-induced internalization of the three deletion mutants (Fig. 3a). In addition, activation by carbachol of endogenously expressed M1 muscarinic acetylcholine receptors in HEK293 cells, which induces PKC- and CaMKII-dependent internalization of mGlu1a (Mundell et al. 2002), produced little if any internalization of the deletion mutants (Fig. 3b), suggesting that the distal C terminus is critical for the PKC- and CaMKII-mediated components of mGlu1a internalization.

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Figure 2. Glutamate-induced internalization of mGlu1a, DM-I, DM-II and DM-III. HEK293 cells were transiently transfected with 5 μg of pcDNA3-mGlu1a, DM-I, DM-II, or DM-III DNA and used in experiments 2 days later. (a) Cells were challenged with glutamate (10 μm; 0–60 min) and surface receptor loss was assessed by ELISA. The data are mean ± SE from six independent experiments. (b) Cells were preincubated with an anti-HA antibody at 4°C for 1 h. Subsequently cells were incubated at 37°C for 30 min in the absence or presence of agonist (glutamate; 10 μm). Receptor localization was determined by immunofluorescence in fixed cells. The mGlu1a and deletion mutant constructs were visualized using a fluorescein-conjugated secondary antibody.

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image

Figure 3. Deletion of the C-terminal tail removes the PKC-dependent component of mGlu1a internalization. (a) Role of PKC in glutamate-induced internalization of mGlu1a, mGlu1b and the deletion mutants. HEK293 cells were transiently transfected with 5 μg of pcDNA3-mGlu1a, mGlu1b, or deletion mutant DNA, and used 2 days later. Cells were treated with the PKC inhibitor GF109203X (2 μm) 15 min before and then during subsequent glutamate addition (10 μm; 30 min). *p < 0.05 compared with respective glutamate-induced cell surface receptor loss in absence of GF109203X (Mann–Whitney U-test). (b) Carbachol promotes marked internalization of mGlu1a (•) but not DM-I (□), DM-II (▵), or DM-III (▿). HEK293 cells were transiently transfected with 5 μg of pcDNA3-mGlu1a or deletion mutant DNA, and used 2 days later. Cells were challenged with carbachol (1 mm; 0–60 min) and surface receptor loss was assessed by ELISA. The data are mean ± SE of five independent experiments.

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A number of recent studies have shown that GRKs are directly involved in the phosphorylation, desensitization and internalization of the mGlu1a receptor (Dale et al. 2000, 2001; Sallese et al. 2000). In agreement with these studies, co-expression of DNM-GRK2 (Lys220Arg) strongly attenuated glutamate-induced internalization of mGlu1a and mGlu1b, with a greater effect on the latter (46% inhibition of mGlu1a and 70% inhibition of mGlu1b; Fig. 4a). With the deletion mutant constructs, DNM-GRK2 co-expression strongly attenuated glutamate-induced internalization of DM-III whilst that of DM-I and II was much less affected (Fig. 4a). The combined effect of PKC inhibition and DNM-GRK2 co-expression was also investigated. In this case, only glutamate-induced mGlu1a internalization was further attenuated by GF109203X treatment in the presence of DNM-GRK2 expression (Fig. 4a). Taken together these observations demonstrate that the distal C terminus of mGlu1a is critical for the PKC-dependent component of internalization, whilst the sequence Ser869–Val893 is critical for GRK-dependent internalization. Further internalization experiments were undertaken following wild-type GRK2 overexpression, which increased the glutamate-induced internalization of all constructs to varying degrees (Fig. 4b). Interestingly however, PKC inhibition no longer significantly reduced glutamate-induced mGlu1a internalization in the presence of overexpressed wild-type GRK2 (surface receptor loss with 10 μm glutamate for 30 min: GRK2: 56 ± 8%; GF109203X + GRK2: 43 ± 5).

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Figure 4. Role of GRK2 and PKC in glutamate-induced internalization of mGlu1a, mGlu1b and the C-terminal tail deletion mutants. (a) HEK293 cells were transiently transfected with 5 μg of either pcDNA3-mGlu1a, mGlu1b, or deletion mutant DNA, along with 5 μg of either pcDNA3 alone (open bars) or pcDNA3-DNM-GRK2 (hatched and filled bars), and used 2 days later. Cells were incubated in the absence or presence (filled bars) of the PKC inhibitor GF109203X (2 μm) for 15 min before and during addition of glutamate (10 μm; 30 min) and surface receptor loss was assessed by ELISA. The data are mean ± SE of five independent experiments. *p < 0.05 compared with respective glutamate-induced cell surface receptor loss in absence of DNM-GRK2 expression or GF109203X addition, **p < 0.05 compared with glutamate-induced cell surface mGlu1a loss in the presence of GRK2-DNM expression (Mann–Whitney U-test). (b) Cells were transiently transfected with 5 μg of either pcDNA3-mGlu1a, mGlu1b or deletion mutant DNA, along with 5 μg of either pcDNA3 alone (open bars) or pcDNA3-GRK2 (hatched bars). Two days later, glutamate-induced (10 μm; 30 min) surface receptor loss was assessed by ELISA. The data are mean ± SE of four independent experiments. Overexpression of DNM-GRK2 (a) or GRK2 (b) > 10-fold over basal as assessed by immunoblot (data not shown).

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Recently we demonstrated that the agonist-induced internalization of mGlu1a is an arrestin- and dynamin-dependent process (Mundell et al. 2001). We therefore determined the role of arrestin and dynamin in internalization of the deletion mutants. Cells were co-transfected with mGlu1a or each of the deletion mutants and either DNM-arr (arrestin-2, 319–418; Krupnick et al. 1997b) or DNM-dyn (dynamin-Lys44Ala; Damke et al. 1994). As shown in Fig. 5, and in agreement with our previous study, expression of either DNM-arr or DNM-dyn strongly inhibited glutamate-induced internalization of mGlu1a. Furthermore, whereas glutamate-induced internalization of all three deletion mutants was inhibited by co-expression of DNM-dyn, DNM-arr only inhibited internalization of DM-III (Fig. 5a). Numerous studies have shown that activation of several different GPCRs leads to rapid recruitment of arrestin-2-GFP and arrestin-3-GFP from cytosol to membrane (Barak et al. 1997; Mundell et al. 2000). We therefore investigated the effects of glutamate on the redistribution of arrestin-2-GFP in cells expressing mGlu1a or each of the three deletion mutants. Prior to glutamate stimulation, all cells displayed a diffuse cytoplasmic distribution of arrestin-2-GFP (Fig. 5b, left-hand panels). Following the addition of 10 μm glutamate, a rapid translocation of arrestin-2-GFP from cytosol to membrane was observed in cells co-expressing either the mGlu1a or DM-III, but not in cells expressing either DM-I or DM-II (Fig. 5b, right-hand panels). Identical results were obtained in cells expressing arrestin-3-GFP (data not shown). It is also interesting to note that subtle shape change often occurred in cells following glutamate addition. Activation by carbachol of endogenously expressed M1 muscarinic acetylcholine receptors in HEK293 cells, which induces PKC- and CaMKII-dependent internalization of mGlu1a (Mundell et al. 2002), also induces arrestin-2-GFP translocation in cells expressing mGlu1a (Fig. 6, top panels; no arrestin-2-GFP translocation was observed following carbachol addition to untransfected cells, data not shown). By contrast, in cells expressing DM-II or DM-III, no carbachol-dependent translocation of arrestin-2-GFP from cytosol to membrane could be observed (Fig. 6, centre middle and lower middle panels), however, in DM-III cells, addition of glutamate after carbachol led to clear arrestin-2-GFP translocation (Fig. 6, lower right panel).

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Figure 5. Glutamate-induced internalization of DM-I and DM-II is arrestin-independent. (a) Effect of DNM-arr or DNM-dyn on glutamate-induced internalization of mGlu1a or deletion mutants DM-I, DM-II and DM-III. HEK293 cells were transiently transfected with 5 μg of either pcDNA3-mGlu1a or deletion mutant DNA, along with 5 μg of either pcDNA3 alone (clear bars), pcDNA3-arrestin-2-(319–418) (DNM-arr, hatched bars) or pcDNA3-dynamin Lys44Ala (DNM-dyn, filled bars). Two days later, cells were challenged with glutamate (10 μm; 30 min) and surface receptor loss was assessed by ELISA. The data are mean ± SE of six independent experiments. *p < 0.05 versus respective pcDNA3 alone (Mann–Whitney U-test). Overexpression of dominant negative mutants > 10-fold over endogenous wild-type proteins as assessed by immunoblot, data not shown. (b) Glutamate-induced translocation of arrestin-2-GFP in HEK293 cells also expressing mGlu1a or DM-III, but not in cells also expressing DM-I or DM-II. Cells grown on poly-l-lysine coverslips were transiently transfected with 5 μg of pcDNA3-mGlu1a or deletion mutant DNA, along with 0.5 μg of arrestin-2-GFP, and used 2 days later. Membrane expression of mGlu1a and the deletion mutants in the cells shown was confirmed by visualization using a rhodamine-conjugated anti-HA antibody (data not shown). Prior to stimulation and viewing, coverslips were mounted in an imaging chamber at 37°C. The initial diffuse cytoplasmic distribution of arrestin-2-GFP is shown prior to agonist stimulation (left panels). Glutamate (10 μm) was added and the redistribution of arrestin-3-GFP was monitored in real time. The images shown in the right-hand panels were collected at 240 s after glutamate addition.

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Figure 6. Muscarinic receptor activation induces translocation of arrestin-2-GFP in cells expressing mGlu1a but not those expressing DM-II or DM-III. HEK293 cells grown on poly-l-lysine coverslips were transiently transfected with 5 μg of pcDNA3-mGlu1a, DM-II, or DM-III DNA, along with 0.5 μg of arrestin-2-GFP. Prior to stimulation and viewing, coverslips were mounted in an imaging chamber at 37°C. The initial diffuse cytoplasmic distribution of arrestin-2-GFP is shown prior to agonist stimulation (left panels). Carbachol (1 mm) was added and any redistribution of arrestin-2-GFP was monitored in real time. Arrestin-2-GFP translocation was only evident in mGlu1a-expressing cells (top middle and top right panels). Where no arrestin translocation was evident following carbachol addition, glutamate was subsequently applied. This now promoted arrestin-2-GFP translocation in DM-III cells (lower right panel) but not in those expressing DM-II (middle right panel). Membrane expression of mGlu1a or deletion mutants in the cells featured was confirmed by visualization using a rhodamine-conjugated anti-HA antibody (data not shown).

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Finally, we also assessed the functional status of the deletion mutants by measuring [3H]IP accumulation following the addition of glutamate (Fig. 7a). Basal IP accumulation was higher in mGlu1a-transfected cells than in those transfected with the deletion mutants (see legend to Fig. 7), probably reflecting the presence of constitutive activity in the former and not the latter. Whereas DM-I was largely uncoupled from IP accumulation, glutamate-stimulated IP accumulation in DM-II- and DM-III-expressing cells was similar to that in mGlu1a-expressing cells (Fig. 7a). We have previously shown that inhibition of second messenger kinases increases agonist-induced mGlu1a signalling (Mundell et al. 2002), presumably by inhibiting a negative feedback mechanism. In the present experiments, IP accumulation following glutamate-induced activation of mGlu1a was enhanced by treatment with the PKC inhibitor GF109203X (2 μm; Fig. 7b). Surprisingly, however, inhibition of PKC also increased DM-II- and DM-III-stimulated IP accumulation, indicating that the PKC mechanism regulating the mGlu1a IP response is not the same as that regulating mGlu1a internalization. Furthermore, because neither PKC activation with phorbol 12-myristate 13-acetate (PMA) nor PKC inhibition with GF109203X significantly altered NaF-stimulated IP accumulation in HEK293 cells (Fig. 7b), it seems likely that the receptor is the main locus of action of PKC. Since mGlu1a appears to undergo arrestin-dependent desensitization in response to glutamate (Mundell et al. 2002), we also determined the arrestin-dependency of desensitization of the deletion mutants. Cells were co-transfected with mGlu1a, DM-II, or DM-III, and arrestin-2. As shown (Fig. 7c), expression of arrestin-2 reduced IP accumulation following glutamate-induced activation of mGlu1a and DM-III, whilst the signalling of DM-II was completely unaffected. This suggests that arrestin interaction with Ser869–Val893 regulates both the desensitization and internalization of mGlu1a. Interestingly, the apparent constitutive activity of mGlu1a was abolished by arrestin-2 overexpression (see legend to Fig. 7).

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Figure 7. Involvement of PKC and arrestins in glutamate-induced inositol phosphate (IP) accumulation in cells expressing mGlu1a (•), DM-I (○), DM-II (▵), or DM-III (▿). HEK293 cells were transiently transfected with either 5 μg of pcDNA3-mGlu1a, DM-I, DM-II, or DM-III DNA, 2 days before assessment of agonist-induced IP accumulation. (a) Total IP levels measured at various times after the addition of 10 μm glutamate. Basal levels of IP accumulation were 3383 ± 659, 2422 ± 794 1792 ± 626 and 1259 ± 563 counts per min (c.p.m.)/well for mGlu1a, DM-I, DM-II and DM-III, respectively. This represented levels of 67 ± 13 (DM-I), 63 ± 11 (DM-II) and 58 ± 13% (DM-III) of those in mGlu1a-transfected cells (taken as 100%). (b) Cells were treated with the PKC inhibitor GF109203X (2 μm) 15 min before and then during addition of glutamate (10 μm; 30 min), and total IP accumulation subsequently measured. Basal levels of IP in GF109203X-untreated cells were 3981 ± 363, 3550 ± 330 and 3194 ± 268 c.p.m./well for mGlu1a, DM-II and DM-III, respectively. This represented levels of 88 ± 3 (DM-II) and 80 ± 4% (DM-III) of those in mGlu1a-transfected cells (taken as 100%). (c) Cells were transiently transfected with receptor construct and 5 μg of either pcDNA3 alone (open bars) or pcDNA3-arrestin-2 (hatched bars). Total IP accumulation was measured after the addition of glutamate (10 μm; 30 min). Basal levels of IP accumulation were 4404 ± 256, 3550 ± 330 and 3194 ± 268 c.p.m for mGlu1a, DM-II and DM-III, respectively, in the absence of arrestin-2 overexpression, and 2901 ± 515, 2877 ± 140 and 2904 ± 112 for mGlu1a, DM-II and DM-III, respectively, in the presence of arrestin-2 overexpression. This represented levels of 59 ± 4 (DM-II) and 77 ± 9% (DM-III) in the absence of arrestin-2 overexpression, and 66 ± 13% (mGlu1a), 65 ± 3 (DM-II) and 66 ± 1% (DM-III) in the presence of arrestin-2 overexpression, of those in mGlu1a-transfected cells in the absence of arrestin-2 overexpression (taken as 100%). In all cases the data are mean ± SE of three to five independent experiments.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Construction of mGlu1a deletion mutants
  6. Cell culture and transfection
  7. Internalization and immunofluorescence microscopy of mGlu1a receptor constructs
  8. Inositol phosphate (IP) determination
  9. Experimental design and statistics
  10. Results
  11. Discussion
  12. Acknowledgement
  13. References

In this study of mGlu1a internalization and signalling we report a number of novel findings. Firstly, the distal C-terminal tail of the receptor (Ser894–Leu1199) is critical for PKC- and CaMKII-dependent regulation of mGlu1a internalization. Secondly, we have identified a region in the proximal C-terminal tail (Ser869–Val893), which is critical for glutamate-stimulated GRK- and arrestin-mediated internalization of mGlu1a. Thirdly, whereas glutamate-induced internalization of mGlu1a is PKC- and GRK2-dependent, that of mGlu1b appears to be driven by GRK2 alone. Finally, even though the distal C-terminal tail plays a critical role in PKC-dependent internalization of mGlu1a, other regions of the receptor (e.g. the second or third intracellular loops) are critical for PKC-dependent mGlu1a desensitization.

To facilitate this study we constructed three C-terminal tail deletion mutants: DM-I, DM-II and DM-III, which lack 352, 331 and 306 amino acids, respectively. Initially we expressed these constructs in HEK293 cells and characterized their ability to undergo glutamate-induced internalization. The deletion mutants, as well as mGlu1a and mGlu1b, were expressed at approximately similar levels at the cell surface, which was somewhat surprising because recent reports (Chan et al. 2001; Dhami et al. 2002) indicate that mGlu1b expresses less well at the surface of HEK293 cells because the Arg-Arg-Lys-Lys endoplasmic reticulum retention motif is present in this splice variant. The reason why we did not also observe this difference is at present unclear, however, our results do allow us to eliminate variable membrane expression levels when considering functional differences between the receptor constructs. In response to glutamate, all three deletion mutants internalized, as assessed by ELISA and immunofluorescent microscopy, but internalization of DM-I and DM-II was significantly impaired as compared with wild-type mGlu1a and DM-III. The main effect of glutamate addition appears to be increased receptor internalization, although we cannot at present exclude the possibility that glutamate addition also affects other components of trafficking such as membrane insertion of new receptor and inhibition of receptor recycling. Interestingly, although only the internalization of mGlu1a and DM-III was inhibited by DNM-arr, the internalization of all constructs was blocked by DNM-dyn. One possibility is that whilst mGlu1a and DM-III internalize via clathrin-coated pits, DM-I and DM-II internalize via a non-clathrin pathway, such as caveolae, which is also dynamin-dependent (Henley et al. 1998). The ability of DM-III to internalize normally indicates that the vast majority of the C-terminal tail of mGlu1a (306 of the proposed 359 amino acid residues in the receptor tail) is not required for efficient glutamate-induced mGlu1a internalization. However, a stretch of 25 amino acids (Ser869–Val893) plays a critical role in promoting optimal trafficking of this receptor.

Recently, the agonist-induced internalization of mGlu1a was shown to be PKC-dependent (Ciruela and McIlhinney 1997; Mundell et al. 2002). In the present study, glutamate-induced internalization of each deletion mutant, as assessed with the PKC inhibitor GF109203X, was PKC-independent. In addition activation by carbachol of endogenously expressed M1 muscarinic acetylcholine receptors in HEK293 cells, which induces marked PKC- and CaMKII-dependent internalization of mGlu1a (Mundell et al. 2002), produced negligible internalization of the deletion mutants. These data indicate that PKC- and CaMKII-dependent internalization of mGlu1a requires the distal C-terminal tail (Ser894–Leu1199) of the receptor. There are a number of putative PKC phosphorylation sites in this region of mGlu1a, and because mGlu1c is directly phosphorylated by PKC (Ciruela et al. 1999), it seems possible that PKC and CaMKII regulation of mGlu1a internalization are mediated by direct phosphorylation of residues in Ser894–Leu1199. Given that DM-I and DM-II are also deletion mutants of mGlu1b, our data further indicate that the PKC- and CaMKII-mediated mGlu1b receptor internalization stimulated by M1 receptor activation depends upon the distal Ser869–Leu906 sequence of the mGlu1b C-terminal tail. Future detailed mutagenesis studies will be required to pin-point which of the distal C-terminal tail residues are required for the PKC/CaMKII components of internalization of mGlu1a and mGlu1b.

A critical role for GRKs in the agonist-induced internalization of mGlu1a has recently been demonstrated (Dale et al. 2000; Sallese et al. 2000). Our study confirms these findings because overexpression of catalytically inactive DNM-GRK2 inhibited the glutamate-induced internalization of mGlu1a. However, it is also clear from our results that GRK2 and PKC together mediate glutamate-induced mGlu1a internalization, as DNM-GRK2 expression plus PKC inhibition was required to abolish effectively internalization. For the deletion mutants, DNM-GRK2 expression strongly inhibited glutamate-induced internalization of DM-III but did not significantly attenuate that of DM-I or DM-II. This suggests that GRK2 phosphorylates within, or interacts with, the stretch of amino acids present in DM-III but not DM-II (i.e. Ser869–Val893). However, analysis of this sequence of amino acids in Fig. 1 reveals no obvious sites for GRK phosphorylation (Ser/Thr residues in the context of the acidic amino acids Asp/Glu). A very recent report indicates that the N terminus of GRK2 can bind to and inhibit the function of mGlu1a in a phosphorylation-independent manner (Dhami et al. 2002). However, this cannot explain the effect on internalization that we observe, because the latter group found DNM-GRK2 to be as effective as wild-type GRK2 in inhibiting mGlu1a function, whereas we find these constructs to have opposite effects on mGlu1a internalization (compare Figs 4a and b), strongly suggesting that GRK2-mediated phosphorylation of mGlu1a is important for internalization. Although DNM-GRK2 did not inhibit glutamate-induced internalization of DM-I or DM-II, wild-type GRK2 expression did increase internalization of these constructs. One possibility is that overexpressed GRK2 forces GRK-dependent internalization by an interaction not normally seen with endogenous levels of GRK2. A similar phenomenon has previously been observed for other GPCRs, such as an internalization-resistant mutant β2-adrenoceptor, for which internalization is restored upon overexpression of GRK2 (Menard et al. 1996). Interestingly, we found the glutamate-induced internalization of the mGlu1b splice variant to be PKC-independent but strongly GRK2-dependent. Previously we found the glutamate-induced desensitization of mGlu1b to be PKC-independent (Mundell et al. 2002), which highlights an important difference in the mechanism of regulation of these two splice variants following glutamate stimulation. However, both mGlu1a and mGlu1b are subject to similar heterologous regulation via PKC and CaMKII following activation of the M1 receptor (Mundell et al. 2002).

The signalling and internalization of numerous GPCRs is now known to be intimately controlled by arrestins, which generally bind to agonist-activated, GRK-phosphorylated GPCRs at the plasma membrane (Goodman et al. 1996). We have previously shown that the glutamate-induced internalization of mGlu1a is arrestin-dependent (Mundell et al. 2001, 2002). For example, agonist-activation of mGlu1a induces arrestin translocation from cytosol to plasma membrane, a phenomenon common to many GPCRs (Barak et al. 1997; Mundell et al. 2000). A specific motif necessary for arrestin–GPCR association has yet to be identified, although arrestins appear to interact with GPCRs that have been phosphorylated by GRKs on S/T residues located in the third intracellular loop or C-terminal tail of the receptor (Ferguson 2001). Of our three deletion mutants, only DM-III underwent glutamate-stimulated arrestin-dependent internalization and promoted arrestin-2-GFP translocation from cytosol to plasma membrane in response to glutamate. In these respects DM-III behaves essentially as wild-type mGlu1a. Hence the same stretch of amino acids critical for GRK-dependent internalization of mGlu1a is also critical for arrestin interaction and arrestin-dependent internalization of this receptor. This region also contains the highly basic Arg-Arg-Lys-Lys sequence, recently identified as a motif critical for neuronal trafficking and targetting of mGlu1 splice variants (Chan et al. 2001; Francesconi and Duvoisin 2002). Whether or not this particular motif is also important for GRK2/arrestin interactions, and whether these functions are related in any way, will be the subject of future experiments.

It is now apparent that arrestins can interact with non-GRK phosphorylated GPCRs (Xiang et al. 2001), and we have recently shown that the agonist-unoccupied mGlu1a undergoes arrestin- and dynamin-dependent internalization following second messenger kinase activation (Mundell et al. 2002). Unlike the full-length mGlu1a, DM-III does not support carbachol-dependent arrestin-2-GFP translocation, probably because of the absence of the PKC component of carbachol-induced internalization in this deletion mutant. It will be interesting to determine whether carbachol-induced PKC activity promotes arrestin binding to the same site as that following glutamate stimulation, or whether a second arrestin interaction site is revealed in the distal C-terminus following PKC activation. Our results indicate that three different kinases, GRK2, PKC and CaMKII regulate mGlu1a activity, and an important question is whether the receptor is a direct substrate for phosphorylation by these kinases. In initial studies we have been unable to detect significant agonist-stimulated mGlu1a phosphorylation following prelabelling of cells with 32P, or by using anti-phosphothreonine and anti-phosphoserine antibodies (data not shown). Indeed, recent studies show that agonist-induced phosphorylation of mGlu1a is not marked (∼1.5-fold increase in mGlu1a phosphorylation when transiently expressed in HEK293 cells; Dale et al. 2000; Dhami et al. 2002). Thus other approaches may be required to quantify agonist-induced mGlu1a phosphorylation satisfactorily.

Our functional coupling studies with mGlu1a and the deletion mutants also uncovered important roles for arrestins and PKC. Initial studies characterizing deletion mutant function via IP accumulation revealed that only DM-II and DM-III functionally coupled to PLC; DM-I is non-functional probably because it lacks the Asp854 residue shown to be critical for efficient mGlu1a/Gq coupling (Pin et al. 1994). Interestingly, although DM-I did not support glutamate-induced IP production, it nevertheless internalized as well as DM-II. It is possible that agonist-induced receptor conformational changes alone are sufficient to trigger internalization of these two deletion mutants, independent of G-protein coupling and second messenger production. Previously we have shown that arrestin-overexpression reduced glutamate-induced mGlu1 splice variant signalling, indicating that arrestins probably play a role in the homologous desensitization of mGlu1a (Mundell et al. 2002). In this study we found that arrestin-2 over-expression attenuated IP production following glutamate stimulation of mGlu1a and DM-III, but not that of DM-II. As with our internalization studies, arrestin-dependent desensitization depended upon Ser869–Val893, and thus arrestin interaction with this region is critical for both desensitization and internalization of mGlu1a. We have previously shown that homologous desensitization of the mGlu1a is partly PKC-dependent (Mundell et al. 2002). Given that PKC does not regulate the internalization of either DM-II or DM-III, we were surprised to find that PKC did regulate the desensitization of these mutants, as PKC inhibition enhanced glutamate-stimulated IP accumulation, as with the wild-type mGlu1a. This indicates that there are two distinct PKC mechanisms that regulate mGlu1a function; one dependent on the distal C terminus that regulates internalization, and one dependent on another part of the receptor that regulates its coupling to IP accumulation. Importantly, IP accumulation in the presence of NaF, which directly activates Gq, was unaffected by PKC inhibition, indicating that the receptor is likely to be the locus of PKC action in the regulation of mGlu1a coupling to IP accumulation. A possibility for the latter could be Thr695 in the second intracellular loop of the receptor, which is probably phosphorylated by PKC and in some model systems appears critical in uncoupling mGlu1a from Gq/11 (Francesconi and Duvoisin, 2000). However, if this is the case then it is unclear why IP accumulation following mGlu1b activation is not also increased following PKC inhibition (Mundell et al. 2002), as it also contains Thr695. Perhaps the C-terminal tail of mGlu1b is able to specifically inhibit the association of PKC with the receptor, or alternatively perhaps the interaction of GRK2 with mGlu1b is of sufficiently high affinity to preclude any effects of PKC activation on mGlu1b desensitization or internalization. Since DM-I and DM-II are not regulated by endogenous GRK2, then PKC may still be able to regulate their activity.

In summary, the present study demonstrates for the first time the crucial importance of different regions of the C-terminal tail of the mGlu1a in the internalization of this receptor. A future goal of our work will be to investigate whether interactions of mGlu1a with GRKs, PKC, CaMKII and arrestins regulate other aspects of receptor function apart from internalization.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Construction of mGlu1a deletion mutants
  6. Cell culture and transfection
  7. Internalization and immunofluorescence microscopy of mGlu1a receptor constructs
  8. Inositol phosphate (IP) determination
  9. Experimental design and statistics
  10. Results
  11. Discussion
  12. Acknowledgement
  13. References
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