Vol. 188, No. 2, 1992 October 30, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 813-819
GLYCINBRGIC LIGANDS MODULATE THE RATE OF PHOSPHORYLATION OF THE GLYCINE RECEPTOR BY PROTEIN RINASE C
Maria-Luisa Centro
Vaello, de Biologia Universidad
Ana Ruiz-Gomez Molecular Autbnoma,
and Federico
Mayor,
Jr.*
"Severe Ochoa" (CSIC-UAM), 28049 Madrid, SPAIN
Received September 17, 1992
Summary. The Q subunit of the glycine receptor purified from rat spinal cord is rapidly and specifically phosphorylated by protein kinase C (Ruiz-Gomez et al., (1991) J. Biol. Chem. 266, 559-566). We report here that the rate of phosphorylation of the glycine receptor by this kinase is higher in the presence of agonists (glycine, p-alanine) than in the presence of antagonists (strychnine, RU-5135). These results suggest that activated glycine receptors would be a preferential target for functional regulation through phosphorylation mechanisms. 0 1992 Academic Press, Inc.
Glycine is an important inhibitory transmitter in the brain stem and spinal cord. The binding of glycine to its receptor promotes a large increase in chloride conductance, which causes membrane hyperpolarization. Such inhibitory actions are selectively antagonized by the alkaloid strychnine (1). Purified glycine receptor (GlyR) preparations from mammalian spinal cord have been reported to contain two glycosylated membrane polypeptides of Mr 48 KDa (a subunit) and 58 KDa (/3 subunit), which form the receptor channel, as well as peripheral proteins associated to cytoplasmic domains of the receptor (2-5). Recent molecular biology studies indicate that the strychnine-sensitive glycine receptor belongs to the receptor-channel superfamily, together with nicotinic acetylcholine, GABAA and some subtypes of glutamate receptors. These ligand-gated ion channels are multimeric structures *To whom correspondence Abbreviations: SDS-PAGE, electrophoresis.
should
GlyR, glycine sodium-dodecyl
be addressed. receptor; sulfate
PKC, protein polyacrylamide
kinase
C; gel
0006-291X/92 $4.00 813
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
composed of homologous subunits which share a common structural motif of four membrane-spanning domains, with a large N-terminal extracellular domain and a large intracellular loop between the third and fourth transmembrane domains (6,7). Recent data suggest that phosphorylation of such intracellular loop by different second messengerstimulated protein kinases may be an important mechanism of modulation of the functionality of these receptors, specially regarding desensitization to their agonists (8,9). Our laboratory has recently reported the "in vitro" phosphorylation of the a subunit of the GlyR by protein kinase of phosphorylation being located in serine 391, c, the site close to the fourth transmembrane domain (10). In the present we have investigated whether occupancy of the GlyR by report, agonists or antagonists -which are known to promote large leading to conformational changes in the receptor protein, channel opening or closing, respectivelyhas any effect on the "in vitro" phosphorylation of purified glycine receptor by protein kinase C. METHODS Protein purification. Protein kinase C was purified from rat brain by sequential chromatography in DE-52, hydroxylapatite and polyacrilamide-phosphatidylserine columns as described receptor was purified from rat spinal cord by (10) * Glycine affinity chromatography on 2-amino-strychnine agarose as previously reported by our laboratory (3). The receptor was specifically eluted from the affinity column with 120 mM KCl, 5 mM EDTA, 5 mM EGTA, 5 mM dithiothreitol, 1 mM benzamidine, 17 milliunits/ ml aprotinin, 2.5 mM iodoacetamide, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM benzetonium chloride, 1% sodium cholate (w/v), 200 mM glycine, 0.18% (w/v) L-aphosphatidylcholine and 25 mM potassium phosphate buffer, pH 7.4, followed by exhaustive dialysis against the elution buffer (except 3 00 mM glycine) to remove the agonist. Glycinedisplaceable strychnine (Amersham) binding to the [ HI purified GlyR was determined as described (3). All operations were carried out at 4*C. Phosphorylation assays. Aliquots of 35 31 of the purified and dialyzed GlyR (aproximately 1 pmol of [ H] strychnine binding sites) were preincubated for 10 min at 4OC in the presence of the desired concentrations of glycinergic agonists (glycine, p-alanine) or antagonists (strychnine and RU-5135, kindly provided by Roussel-Uclaf Laboratories), and subsequently incubated at 30pC for different periods of time with 1.10-4 units of protein kinase C under phosphorylating conditions exactly as described (10). Reactions were stopped by the addition of SDS-PAGE sample buffer. Radiolabeled proteins were separated by SDS-PAGE (10% acrylamide) and visualized by autoradiography. The phosphorylation of the a subunit of the 814
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
GlyR was quantitated by scanning the autoradiograms using a Molecular Dynamics 300 A computing densitometer. In some experiments, the radioactive content of the a subunit band excised from the dried gel was determined by Cerenkov and the phosphorylation stoichiometry calculated spectroscopy, as reported (10). RESULTS AND DISCUSSION Figures IA and 1B show the phosphorylation of the Q subunit of the purified GlyR after 15 min and 2 min of incubation with protein kinase C, respectively, either in the presence of the agonist glycine (Gly) or the specific antagonist strychnine (Str) at saturating concentrations. Whereas no apparent differences in phosphorylation are detected in the 15 min assay (or at longer times of incubation, data not shown) a higher phosphorylation of the a subunit in the presence of glycine is clearly detected at the shorter time of incubation. Together with our previous experiments showing GlyR phosphorylation in the absence of ligands (lo), these results suggest that glycinergic ligands may modulate the rate of phosphorylation of the GlyR by protein kinase C, without affecting the extent of the "in vitro" phosphorylation of the
c -rPUG P
GlyR
str
G)Y 2
3 0.5 1 2
3 (min)
-PUG
D-
-
15 min
GlyR
2 min
-
PKC
-
GlyR
fi, &b V-J
l,*fV
n
\i
Figure 1. Effect of the presence of glycine or strychnine on the "in vitro*' phosphorylation of the a subunit of the Glycine by protein kinase C. Receptor Purified and dialyzed GlyR was preincubated for 10 min at dpC with 500 @4 glycine (Gly) or 50 PM strychnine (Str) before adding purified protein kinase C and phosphorylation buffer as detailed in Methods. Incubations lasted 15 min (panel A), 2 min (panel B) or 0.5 to 3 min as indicated in each lane (panel proteins were resolved by SDS-PAGE and Cl * Radiolabeled autoradiography. The migration of the visualized by autophosphorylation band of protein kinase C (PKC) and of the a subunit of the GlyR is marked with arrows. The lower part of panel C shows an analysis of the labeling intensity of the a subunit band with an scanning laser densitometer. The gels shown are representative of four independent experiments.
815
Vol.
188,
No.
2,
1992
R-Ala RU A --
BIOCHEMICAI
B
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1co
Figure 2. Effect of the presence of p-alanine the "in vitro" phosphorylation of the a subunit Receptor by protein kinase C. Comparison with other glycinergic ligands.
or RU-5135 on of the Glycine the effects of
Panel A. Purified and dialyzed GlyR was preincubated with 500 I.~M p-alanine (P-Ala) or 50 PM RU-5135 (RU) before adding purified protein kinase C and phosphorylation buffer as detailed in Methods. Incubations lasted l-3 min as indicated in each lane. Radiolabeled proteins were resolved by SDS-PAGE and visualized by autoradiography. The position of protein kinase C (PRC) and the a subunit of the GlyR is indicated. The gel shown is representative of three independent experiments. Panel B. Comparison of the effect of different glycinergic ligands on short-term GlyR phosphorylation by PKC. The labeling intensity of the a subunit of the GlyR after 3 min of incubation with protein kinase C was measured by laser densitometry and normalized with respect to the value attained in the presence of glycine which was taken as 100%. Agonists glycine and P-alanine were tested at 500 pM and the antagonists at 50 @i. Results are mean of three experiments (SEM less than 15%).
explore this point, a more detailed study of the effects of agonist or antagonist occupancy on the initial rates of a subunit phosphorylation by protein kinase C were performed. Fig. 1C confirms that the 48 KDa a subunit is more rapidly phosphorylated in the presence of the agonist glycine (open channel conformation) than when interacting with the antagonist strychnine (closed channel conformation). In the presence of glycine the calculated initial rate of phosphorylation is approximately a-fold of that found with strychnine. Similar results are obtained (Fig. 2A) when comparing the effects of p-alanine, a full agonist of the GlyR an steroid derivative which is able to block (1) I and RU-5135, both the GABAA and the glycine receptor channels (11). Figure 2B show that, after 3 min of incubation with protein kinase C, GlyR a subunits are phosphorylated to the same extent in the presence of 500 /.LM glycine or p-alanine (a stoichiometry of phosphorylation of 0.2-0.3 pmol of phosphate incorporated per pmol of a subunit being usually attained), whereas a similar percentage of "inhibition11 is detected with saturating receptor.
To further
816
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
of strychnine or RU-5135. Such effects of concentrations glycinergic ligands are dose-dependent (data not shown). It is worth noting that the autophosporylation of protein kinase C is not affected by the presence of glycinergic ligands. In some experiments, the phosphorylation of a band of 58 KDa seem also to proceed more rapidly in the aproximately presence of agonists (see Fig. 1C). This band could correspond to the B subunit of the GlyR, which sequence has been reported to contain potential phosphorylation sites for protein kinase c (12). In summary, our data suggest that the Q subunit of the GlyR would be more readily regulated by protein kinase C when occupied by an agonist, i.e., in the open channel conformation. Receptor activation has been shown to be required for the phosphorylation of G protein-coupled receptors by specific protein kinases (13, 14), and padrenergic receptor agonists have been reported to increase the rate of phosphorylation of its receptor by CAMP-dependent protein kinase (PKA) (15, 16). More recently, it has been described that the "in vitro" phosphorylation of a putative subunit of the chick cerebellar kainate receptor is modulated by kainatergic ligands (17). Given the key conformational and functional changes that agonists promote in plasma membrane receptors, it is not surprising to find agonist-promoted changes in the interaction of receptors with their regulatory proteins. Conversely, we have also investigated whether prior phosphorylation of GlyR by protein kinase C affects the subsequent interaction of glycine, measured by the ability to displace [3H]-strychnine binding to the purified receptor. Purified and dialyzed GlyR preparations were incubated for 30 min at 30pC with protein kinase C as described (lo), either in the presence of ATP (phosphorylating conditions) or the nonhydrolizable analog AMP-PNP (control conditions). Under control conditions glycine displaced [3H]-strychnine binding with an IC50 of 5828 I.LM and nH of 0.55 + 0.1 (n=3, Similar to values previously reported whereas for the (3)), phosphorylated GlyR the IC50 and nH values were 200 + 54 PM and 0.45 + 0.05, respectively. Thus, our data indicate a 3fold decrease in the potency of glycine to interact with the GlyR when this protein is phosphorylated by protein kinase C. our results are consistent with a role Taken together, for protein kinase C in the mechanisms of GlyR regulation and 817
Vol.
188,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
desensitization. Activated receptors would be more easily phosphorylated by protein kinase C which, in turn, would lead to altered glycine binding and decreased glycine-mediated chloride currents (M.L. Vaello et al., manuscript in preparation). The phosphorylation of other receptors of the and same superfamily, such as the nicotinic acetylcholine GABAA receptors has also been shown to modulate receptor function and desensitization (8,9, 18). Although further cellular and functional studies are required, it is tempting to suggest the existence of a close relationship between GlyR activation and functional regulation. Messengers regulating protein kinase C activity could affect the neuronal response to glycine, which would in turn modulate the rate of regulation of its own receptor. Such mechanisms would help to integrate different extracellular signals at the neuronal level, and account for transient changes in the efficiacy of synaptic transmission. Acknowledgments. This work was supported by CICYT PB870216, Boehringer Ingelheim and an institutional grant from Fundacion Ramon Areces. M.V. holds a Ministerio de Eduacion y Ciencia Fellowship. The authors thank Prof. F. Mayor for continuous encouragement, and M. Sanz for expert secretarial assistance. REFERENCES 1.
Aprison, 203-295.
2.
Pfeiffer, Chem.,
M.H.
257,
and
Daly,
F., Graham, 9389-9393.
E.C. D.
(1978)
and
Bets,
3.
Garcia-Calvo, M., Ruiz-Gomez, A., Valdivieso, F. and Mayor, Jr., 28, 6405-6409.
4.
Langosch, D., 298, 113-117.
5.
Prior, P., Schmitt, B., Multhaup, G., Beyreuther, Langosch, D., Kirsch, J. 1161-1170.
6.
Barnard, E-A., Trends Neurosci.,
7.
Betz,
8.
Huganir, 567.
H.
(1990). R.L.
Hoch,
W.
and
and
H.
Neurochem. (1982)
J.
3, Biol.
Vazquez, J., Morato, E., F. (1989) Biochemistry,
Betz,
H.
(1992)
Grenningloh, K., Maulet, and Betz, H.
Darlison, M.G. 10, 502-509. Neuron.,
Adv.
and
FEBS Lett.,
G., Pribilla, Y., Werner, (1992) Neuron.,
Seeburg,
P.
I., P., 8,
(1987).
5, 383-392.
Greengard, 818
P.
(1990).
Neuron.,
5,
555-
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Blackstone, Moss, S.J., FASEB J. 6, 2514-2523.
9.
Swope, S.L., R.L. (1992).
10.
Ruiz-Gomez, A., Jr., F. (1991).
Vaello, J. Biol.
11.
Curtis, 383-384.
Malik,
12.
Grenningloh, Beyreuther, 963-970.
13.
Benovic, J.L., and Lefkowitz,
14.
Dohlman, H.G., Thorner, J., Caron, R.J. (1991) Annu. Rev. Biochem., 60,
15.
Benovic, J-L., C ., Yoshimasa, R.J. (1985) J.
16.
Walaas, S.I. 43, 229-349.
17.
Ortega, A. 21404-21406.
18.
Moss, S.J., R.L. (1992)
D. and
M.L., Chem., R.
C.D.
Valdivieso, F. 266, 559-566.
(1985)
Eur.
J.
G., Pribilla, L., Prior, K., Taleb, 0. and Betz, H. Mayor, Jr., R.J. (1986)
Pike, L.J., T., Codina, Biol. Chem., and
and Teichberg, Smart, Science,
T.G., 257,
V.
Blackstone, 661-665.
819
Mayor, 110,
R-L., Caron, 869-872.
M.C. and 653-688.
(1991).
(1990).
and
Pharmacol.,
Cerione, R.A., J., Caron, M.G. 260, 7094-7104. P.
Huganir,
P., Multhaup, (1990). Neuron.,
F., Somers, Nature, 322,
Greengard,
and
J.
C.D.
M.G.
Lefkowitz,
Staniszewski, and Lefkowitz,
Pharmacol., Biol.
G., 4,
Rev.
Chem., and
265,
Huganir,
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 813-819
GLYCINBRGIC LIGANDS MODULATE THE RATE OF PHOSPHORYLATION OF THE GLYCINE RECEPTOR BY PROTEIN RINASE C
Maria-Luisa Centro
Vaello, de Biologia Universidad
Ana Ruiz-Gomez Molecular Autbnoma,
and Federico
Mayor,
Jr.*
"Severe Ochoa" (CSIC-UAM), 28049 Madrid, SPAIN
Received September 17, 1992
Summary. The Q subunit of the glycine receptor purified from rat spinal cord is rapidly and specifically phosphorylated by protein kinase C (Ruiz-Gomez et al., (1991) J. Biol. Chem. 266, 559-566). We report here that the rate of phosphorylation of the glycine receptor by this kinase is higher in the presence of agonists (glycine, p-alanine) than in the presence of antagonists (strychnine, RU-5135). These results suggest that activated glycine receptors would be a preferential target for functional regulation through phosphorylation mechanisms. 0 1992 Academic Press, Inc.
Glycine is an important inhibitory transmitter in the brain stem and spinal cord. The binding of glycine to its receptor promotes a large increase in chloride conductance, which causes membrane hyperpolarization. Such inhibitory actions are selectively antagonized by the alkaloid strychnine (1). Purified glycine receptor (GlyR) preparations from mammalian spinal cord have been reported to contain two glycosylated membrane polypeptides of Mr 48 KDa (a subunit) and 58 KDa (/3 subunit), which form the receptor channel, as well as peripheral proteins associated to cytoplasmic domains of the receptor (2-5). Recent molecular biology studies indicate that the strychnine-sensitive glycine receptor belongs to the receptor-channel superfamily, together with nicotinic acetylcholine, GABAA and some subtypes of glutamate receptors. These ligand-gated ion channels are multimeric structures *To whom correspondence Abbreviations: SDS-PAGE, electrophoresis.
should
GlyR, glycine sodium-dodecyl
be addressed. receptor; sulfate
PKC, protein polyacrylamide
kinase
C; gel
0006-291X/92 $4.00 813
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
composed of homologous subunits which share a common structural motif of four membrane-spanning domains, with a large N-terminal extracellular domain and a large intracellular loop between the third and fourth transmembrane domains (6,7). Recent data suggest that phosphorylation of such intracellular loop by different second messengerstimulated protein kinases may be an important mechanism of modulation of the functionality of these receptors, specially regarding desensitization to their agonists (8,9). Our laboratory has recently reported the "in vitro" phosphorylation of the a subunit of the GlyR by protein kinase of phosphorylation being located in serine 391, c, the site close to the fourth transmembrane domain (10). In the present we have investigated whether occupancy of the GlyR by report, agonists or antagonists -which are known to promote large leading to conformational changes in the receptor protein, channel opening or closing, respectivelyhas any effect on the "in vitro" phosphorylation of purified glycine receptor by protein kinase C. METHODS Protein purification. Protein kinase C was purified from rat brain by sequential chromatography in DE-52, hydroxylapatite and polyacrilamide-phosphatidylserine columns as described receptor was purified from rat spinal cord by (10) * Glycine affinity chromatography on 2-amino-strychnine agarose as previously reported by our laboratory (3). The receptor was specifically eluted from the affinity column with 120 mM KCl, 5 mM EDTA, 5 mM EGTA, 5 mM dithiothreitol, 1 mM benzamidine, 17 milliunits/ ml aprotinin, 2.5 mM iodoacetamide, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM benzetonium chloride, 1% sodium cholate (w/v), 200 mM glycine, 0.18% (w/v) L-aphosphatidylcholine and 25 mM potassium phosphate buffer, pH 7.4, followed by exhaustive dialysis against the elution buffer (except 3 00 mM glycine) to remove the agonist. Glycinedisplaceable strychnine (Amersham) binding to the [ HI purified GlyR was determined as described (3). All operations were carried out at 4*C. Phosphorylation assays. Aliquots of 35 31 of the purified and dialyzed GlyR (aproximately 1 pmol of [ H] strychnine binding sites) were preincubated for 10 min at 4OC in the presence of the desired concentrations of glycinergic agonists (glycine, p-alanine) or antagonists (strychnine and RU-5135, kindly provided by Roussel-Uclaf Laboratories), and subsequently incubated at 30pC for different periods of time with 1.10-4 units of protein kinase C under phosphorylating conditions exactly as described (10). Reactions were stopped by the addition of SDS-PAGE sample buffer. Radiolabeled proteins were separated by SDS-PAGE (10% acrylamide) and visualized by autoradiography. The phosphorylation of the a subunit of the 814
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
GlyR was quantitated by scanning the autoradiograms using a Molecular Dynamics 300 A computing densitometer. In some experiments, the radioactive content of the a subunit band excised from the dried gel was determined by Cerenkov and the phosphorylation stoichiometry calculated spectroscopy, as reported (10). RESULTS AND DISCUSSION Figures IA and 1B show the phosphorylation of the Q subunit of the purified GlyR after 15 min and 2 min of incubation with protein kinase C, respectively, either in the presence of the agonist glycine (Gly) or the specific antagonist strychnine (Str) at saturating concentrations. Whereas no apparent differences in phosphorylation are detected in the 15 min assay (or at longer times of incubation, data not shown) a higher phosphorylation of the a subunit in the presence of glycine is clearly detected at the shorter time of incubation. Together with our previous experiments showing GlyR phosphorylation in the absence of ligands (lo), these results suggest that glycinergic ligands may modulate the rate of phosphorylation of the GlyR by protein kinase C, without affecting the extent of the "in vitro" phosphorylation of the
c -rPUG P
GlyR
str
G)Y 2
3 0.5 1 2
3 (min)
-PUG
D-
-
15 min
GlyR
2 min
-
PKC
-
GlyR
fi, &b V-J
l,*fV
n
\i
Figure 1. Effect of the presence of glycine or strychnine on the "in vitro*' phosphorylation of the a subunit of the Glycine by protein kinase C. Receptor Purified and dialyzed GlyR was preincubated for 10 min at dpC with 500 @4 glycine (Gly) or 50 PM strychnine (Str) before adding purified protein kinase C and phosphorylation buffer as detailed in Methods. Incubations lasted 15 min (panel A), 2 min (panel B) or 0.5 to 3 min as indicated in each lane (panel proteins were resolved by SDS-PAGE and Cl * Radiolabeled autoradiography. The migration of the visualized by autophosphorylation band of protein kinase C (PKC) and of the a subunit of the GlyR is marked with arrows. The lower part of panel C shows an analysis of the labeling intensity of the a subunit band with an scanning laser densitometer. The gels shown are representative of four independent experiments.
815
Vol.
188,
No.
2,
1992
R-Ala RU A --
BIOCHEMICAI
B
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1co
Figure 2. Effect of the presence of p-alanine the "in vitro" phosphorylation of the a subunit Receptor by protein kinase C. Comparison with other glycinergic ligands.
or RU-5135 on of the Glycine the effects of
Panel A. Purified and dialyzed GlyR was preincubated with 500 I.~M p-alanine (P-Ala) or 50 PM RU-5135 (RU) before adding purified protein kinase C and phosphorylation buffer as detailed in Methods. Incubations lasted l-3 min as indicated in each lane. Radiolabeled proteins were resolved by SDS-PAGE and visualized by autoradiography. The position of protein kinase C (PRC) and the a subunit of the GlyR is indicated. The gel shown is representative of three independent experiments. Panel B. Comparison of the effect of different glycinergic ligands on short-term GlyR phosphorylation by PKC. The labeling intensity of the a subunit of the GlyR after 3 min of incubation with protein kinase C was measured by laser densitometry and normalized with respect to the value attained in the presence of glycine which was taken as 100%. Agonists glycine and P-alanine were tested at 500 pM and the antagonists at 50 @i. Results are mean of three experiments (SEM less than 15%).
explore this point, a more detailed study of the effects of agonist or antagonist occupancy on the initial rates of a subunit phosphorylation by protein kinase C were performed. Fig. 1C confirms that the 48 KDa a subunit is more rapidly phosphorylated in the presence of the agonist glycine (open channel conformation) than when interacting with the antagonist strychnine (closed channel conformation). In the presence of glycine the calculated initial rate of phosphorylation is approximately a-fold of that found with strychnine. Similar results are obtained (Fig. 2A) when comparing the effects of p-alanine, a full agonist of the GlyR an steroid derivative which is able to block (1) I and RU-5135, both the GABAA and the glycine receptor channels (11). Figure 2B show that, after 3 min of incubation with protein kinase C, GlyR a subunits are phosphorylated to the same extent in the presence of 500 /.LM glycine or p-alanine (a stoichiometry of phosphorylation of 0.2-0.3 pmol of phosphate incorporated per pmol of a subunit being usually attained), whereas a similar percentage of "inhibition11 is detected with saturating receptor.
To further
816
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
of strychnine or RU-5135. Such effects of concentrations glycinergic ligands are dose-dependent (data not shown). It is worth noting that the autophosporylation of protein kinase C is not affected by the presence of glycinergic ligands. In some experiments, the phosphorylation of a band of 58 KDa seem also to proceed more rapidly in the aproximately presence of agonists (see Fig. 1C). This band could correspond to the B subunit of the GlyR, which sequence has been reported to contain potential phosphorylation sites for protein kinase c (12). In summary, our data suggest that the Q subunit of the GlyR would be more readily regulated by protein kinase C when occupied by an agonist, i.e., in the open channel conformation. Receptor activation has been shown to be required for the phosphorylation of G protein-coupled receptors by specific protein kinases (13, 14), and padrenergic receptor agonists have been reported to increase the rate of phosphorylation of its receptor by CAMP-dependent protein kinase (PKA) (15, 16). More recently, it has been described that the "in vitro" phosphorylation of a putative subunit of the chick cerebellar kainate receptor is modulated by kainatergic ligands (17). Given the key conformational and functional changes that agonists promote in plasma membrane receptors, it is not surprising to find agonist-promoted changes in the interaction of receptors with their regulatory proteins. Conversely, we have also investigated whether prior phosphorylation of GlyR by protein kinase C affects the subsequent interaction of glycine, measured by the ability to displace [3H]-strychnine binding to the purified receptor. Purified and dialyzed GlyR preparations were incubated for 30 min at 30pC with protein kinase C as described (lo), either in the presence of ATP (phosphorylating conditions) or the nonhydrolizable analog AMP-PNP (control conditions). Under control conditions glycine displaced [3H]-strychnine binding with an IC50 of 5828 I.LM and nH of 0.55 + 0.1 (n=3, Similar to values previously reported whereas for the (3)), phosphorylated GlyR the IC50 and nH values were 200 + 54 PM and 0.45 + 0.05, respectively. Thus, our data indicate a 3fold decrease in the potency of glycine to interact with the GlyR when this protein is phosphorylated by protein kinase C. our results are consistent with a role Taken together, for protein kinase C in the mechanisms of GlyR regulation and 817
Vol.
188,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
desensitization. Activated receptors would be more easily phosphorylated by protein kinase C which, in turn, would lead to altered glycine binding and decreased glycine-mediated chloride currents (M.L. Vaello et al., manuscript in preparation). The phosphorylation of other receptors of the and same superfamily, such as the nicotinic acetylcholine GABAA receptors has also been shown to modulate receptor function and desensitization (8,9, 18). Although further cellular and functional studies are required, it is tempting to suggest the existence of a close relationship between GlyR activation and functional regulation. Messengers regulating protein kinase C activity could affect the neuronal response to glycine, which would in turn modulate the rate of regulation of its own receptor. Such mechanisms would help to integrate different extracellular signals at the neuronal level, and account for transient changes in the efficiacy of synaptic transmission. Acknowledgments. This work was supported by CICYT PB870216, Boehringer Ingelheim and an institutional grant from Fundacion Ramon Areces. M.V. holds a Ministerio de Eduacion y Ciencia Fellowship. The authors thank Prof. F. Mayor for continuous encouragement, and M. Sanz for expert secretarial assistance. REFERENCES 1.
Aprison, 203-295.
2.
Pfeiffer, Chem.,
M.H.
257,
and
Daly,
F., Graham, 9389-9393.
E.C. D.
(1978)
and
Bets,
3.
Garcia-Calvo, M., Ruiz-Gomez, A., Valdivieso, F. and Mayor, Jr., 28, 6405-6409.
4.
Langosch, D., 298, 113-117.
5.
Prior, P., Schmitt, B., Multhaup, G., Beyreuther, Langosch, D., Kirsch, J. 1161-1170.
6.
Barnard, E-A., Trends Neurosci.,
7.
Betz,
8.
Huganir, 567.
H.
(1990). R.L.
Hoch,
W.
and
and
H.
Neurochem. (1982)
J.
3, Biol.
Vazquez, J., Morato, E., F. (1989) Biochemistry,
Betz,
H.
(1992)
Grenningloh, K., Maulet, and Betz, H.
Darlison, M.G. 10, 502-509. Neuron.,
Adv.
and
FEBS Lett.,
G., Pribilla, Y., Werner, (1992) Neuron.,
Seeburg,
P.
I., P., 8,
(1987).
5, 383-392.
Greengard, 818
P.
(1990).
Neuron.,
5,
555-
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Blackstone, Moss, S.J., FASEB J. 6, 2514-2523.
9.
Swope, S.L., R.L. (1992).
10.
Ruiz-Gomez, A., Jr., F. (1991).
Vaello, J. Biol.
11.
Curtis, 383-384.
Malik,
12.
Grenningloh, Beyreuther, 963-970.
13.
Benovic, J.L., and Lefkowitz,
14.
Dohlman, H.G., Thorner, J., Caron, R.J. (1991) Annu. Rev. Biochem., 60,
15.
Benovic, J-L., C ., Yoshimasa, R.J. (1985) J.
16.
Walaas, S.I. 43, 229-349.
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