Chem. Senses 24: 295-299,
1999
© Oxford University Press
Protein Kinase Cß and 
Selectively Phosphorylate Odorant and Metabotropic Glutamate Receptors
Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA
Correspondence to be sent to: Richard C. Bruch, Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA. e-mail:rbruch{at}lsuvm.sncc.lsu.edu
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Abstract |
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 Top Abstract IntroductionMethods, results and discussionReferences |
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Recombinant protein segments from a metabotropic glutamate receptor and from an odorant receptor were used as substrates in protein kinase C phosphorylation assays. Protein kinase Cß and
phosphorylated an intracellular consensus phosphorylation site in the metabotropic
glutamate
receptor. Only protein kinase C phosphorylated a novel extracellular consensus
phosphorylation
site
in the odorant receptor. These results suggest differential regulation of these receptors by protein
kinase
C isotypes. |
Introduction |
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TopAbstractIntroductionMethods, results and discussionReferences |
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It is well established that seven transmembrane domain (7TMD) receptors are regulated by protein kinases. Protein kinase-mediated phosphorylation of 7TMD receptors initiates a series of molecular events leading to desensitization and internalization of the receptors (reviewed in Bohmet al., 1997;
Ferguson et al., 1997). Most
7TMD
receptors are phosphorylated in a ligand-dependent manner by Gprotein-coupled receptor kinases
(GRKs). We (Bruch et al., 1997) and others (Dawson et al., 1993) have shown that GRK3 (ßARK2) is expressed in
vertebrate
olfactory neurons. Targeted disruption of the GRK3 gene in transgenic knockout mice led to loss
of
odorant-induced desensitization, indicating a critical role for GRK3 in odorant receptor
desensitization (Peppel et al.,, 1997). In addition to GRKs,
7TMD receptors may also
be phosphorylated in a ligand independent manner by second messenger-dependent protein
kinases
such as the cyclic AMP-dependent protein kinase and protein kinase C (PKC). The PKC family
consists of a dozen genes and splice variants (Liu, 1996). Two PKC
family
members, ß and , are expressed in catfish olfactory receptor neurons (Bruch et
al., 1997).
Channel catfish (Ictalurus punctatus) olfactory receptor neurons express multiple
7TMD
receptors such as odorant receptors (Ngai et al., 1993) and
metabotropic glutamate receptors (mGluRs; Medler et al., 1998).
Inspection of the originally published (Ngai et al., 1993) catfish
odorant receptor sequences indicates that half of the receptors contain a PKC consensus
phosphorylation site located between transmembrane domains IV and V. Based on current
understanding of 7TMD receptor structure and membrane topology, this region of the odorant
receptors containing the consensus site is predicted to be extracellular loop 2. The presence of an
extracellular PKC consensus site in these receptors is an apparently novel feature of their
structures and
may be a unique structural feature within the 7TMD receptor superfamily. In contrast, mGluR1,
which
is also expressed in catfish olfactory receptor neurons (Medler et al., 1998), contains a PKC consensus site between transmembrane domains III and IV. This
region
of mGluR1 is predicted to be intracellular loop 2.
Given the different positions and predicted membrane orientation of PKC phosphorylation
sites in
odorant receptors and mGluRs, we investigated the ability of PKCß and , previously
shown
to
be expressed in catfish olfactory receptor neurons (Bruch et al., 1997), to phosphorylate the consensus sequences in both receptors in vitro.
Segments of the
receptors containing the PKC consensus sequences were expressed as fusion proteins, purified
and
used as substrates in phosphorylation assays to determine whether PKC ß and
displayed
differential substrate specificities and whether PKC would phosphorylate the unusual odorant
receptor
extracellular consensus site. Both PKC isotypes phosphorylated the intracellular mGluR1
consensus
site. In contrast, only PKC phosphorylated the extracellular odorant receptor consensus
site. The
specificity of the phosphorylation assay was confirmed using mutant (S/T to A) fusion proteins
that
were not phosphorylated by either PKC isotype. These results show that PKC isotypes are
capable of
differential interaction with odorant receptors and mGluRs in vitro and suggest these
receptors
may be differentially regulated by PKC isotypes.
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Methods, results and discussion |
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TopAbstractIntroductionMethods, results and discussionReferences |
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Wild type and mutant hydrophilic loops of the odorant receptor and mGluR1 containing the consensus PKC phosphorylation sequences were amplified from previously cloned receptor PCR products (Medler et al., 1998
) using specific primers for each
receptor (Figure 1). Receptor segments were amplified with Taq
polymerase using the
following thermal cycling program: 90°C for 5 min, 95°C for 1 min, 48°C (or
54°C for mGluR1) for 1 min, and 72°C for 1 min for 40 cycles. After a final 10 min
extension at 72°C, the reactions were terminated by heating to 95°C for 15 min and
cooling
of the samples to 4°C over several minutes. Amplified PCR products (~150 bp for the
odorant
receptor and 100 bp for mGluR1) were gel purified and ligated into pCR 2.1 (Invitrogen,
Carlsbad,
CA). Complete DNA sequence analysis on both strands confirmed that the products encoded the
desired hydrophilic loops of the receptors (Figure 1).
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Site-directed mutagenesis was used to mutate the serine and threonine residues within the consensus PKC sequences to alanine. Mutagenesis was performed using the Quikchange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). Specific primers for each receptor were used to obtain the desired mutations (Figure 1). The primers were used in the following thermal cycling program: 95°C for 1 min, 55°C for 1 min and 68°C for 12 min for 1 cycle followed by 15 cycles of the same program with the 95°C denaturation shortened to 30 s. Products were digested with DpnI to remove non-mutated plasmids and transformed into Epicurean Coli XL1-Blue Supercompetent cells. Purified plasmids with inserts were sequenced to confirm mutagenesis of the desired nucleotide and to verify that no additional mutations were introduced by PCR.
Wild type and mutant receptor sequences in pCR2.1 were excised with EcoRI and subcloned into the pThioHis prokaryotic expression vector (Invitrogen). DNA sequence analysis confirmed the correct in frame orientation of the subcloned sequences. Protein expression was induced with 1 mM IPTG at 37°C and proceeded for 2 h. Because the thioredoxin moiety in the fusion protein localizes to osmotically sensitive adhesion zones, purification of the fusion proteins was performed by osmotic shock following a protocol provided by Invitrogen in the presence of 0.1 mg/ml PMSF. Proteins isolated by osmotic shock were separated on a 15% SDS–polyacrylamide gel. Fusion proteins were located by Western blotting with the antiThio antibody (Invitrogen, 1:5000 dilution). The fusion proteins were excised from the gel, electroeluted and concentrated in Centricon-10 devices (Millipore, Bedford, MA). Due to a potential phosphorylation site in the fusion proteins upstream from the receptor sequences, enterokinase (Invitrogen) was used to cleave the fusion proteins. Following overnight incubation with 1 U of the enzyme at 4°C, the reaction mixtures were separated on an 18% SDS–polyacrylamide gel. Proteins were visualized with GELCODE Blue Stain Reagent (Pierce, Rockford, IL) and bands of the appropriate size (~9 kDa for the odorant receptor and 5.5 kDa for mGluR1) were excised, electroeluted, concentrated with Centricon-3 devices and exchanged into assay dilution buffer (20 mM MOPS, pH 7.2, 1 mM dithiothreitol, 1 mM CaCl 2).
A mixed micellar assay was used to evaluate PKC phosphorylation (Hannunet al., 1985).
Purified (>95%) recombinant PKCßII and (Upstate Biotechnology, Lake Placid,
NY)
were
diluted tenfold with enzyme dilution buffer (supplied by the vendor). [
-32P]ATP
(3000 Ci/mmol) was diluted tenfold with assay dilution buffer supplemented with 75 mM MgCl 2 and 500 µM ATP. Dioleoyl L-
-phosphatidyl-L-serine
(PS) and 1-oleoyl-2-acetyl-sn-glycerol (OAG) were diluted in chloroform and dried in a
glass
tube. The lipids were solubilized in 3% Triton X-100 in assay dilution buffer to final
concentrations of
0.5 mg/ml PS and 0.05 mg/ml OAG. Assays contained ~400 ng of homogeneous substrate, 10
µl of lipids and 10 µl of diluted radiolabeled ATP in a total volume of 50 µl.
Reactions were initiated by the addition of PKC (25 ng) and were incubated at room temperature
for
25 min. Parallel samples lacking substrate were used to monitor PKC autophosphorylation.
Reactions
were stopped with 3 vol of 10% TCA or with 1 vol of 2x Laemmli buffer. Samples
precipitated
with TCA were analyzed in a filter assay (Bruch et al., 1997).
Samples in Laemmli buffer were separated on a 15% SDS–polyacrylamide gel. The dry
gels
were exposed to X-ray film at -80°C for at least 16 h.
The PKC phosphorylation sequences identified in the odorant receptor and in mGluR1 (Figure 1) conformed to the generalized consensus sequence (R/K)1–3-(X ) 0–2-S/T(X) 0–2-(R/K)1–3 where X represents any amino acid (Pinna and Ruzzene, 1996). The
hydrophilic loops from both receptors containing the PKC consensus sequences were expressed
as
fusion proteins and used as substrates in phosphorylation assays. To verify that the PKC isotypes
were
active, assays were performed using histone (type III, Sigma Chemical Co., St Louis, MO), a
generic
PKC substrate. Enzyme activity was determined by measuring the incorporation of radiolabeled
phosphate into TCA precipitable material. In triplicate assays, PKCß had a sp. act. of 0.124
± 0.017 nmol phosphate/µg histone/min while that of PKC was 0.196
±
0.014 nmol phosphate/µg histone/ min. These results confirmed that both PKC enzymes
were
catalytically active. Receptor hydrophilic loops were also PKC substrates. Both PKCß and
phosphorylated the mGluR1 loop; however, only PKC was able to phosphorylate the
odorant
receptor loop (Figure 2). When mutant receptor loops were used as
substrates
no phosphorylation was detected, thereby confirming the specificity of the assay.
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This study investigated in vitro the specific interactions between the PKC isotypes and 7TMD receptors expressed in olfactory receptor neurons. We have previously shown that the calcium-sensitive PKCß and the calcium-insensitive PKC
are expressed in olfactory
neurons.
We also showed that a specific inhibitor of calcium-sensitive PKC isotypes reduced
odorant-induced
phosphorylation to basal levels in isolated cilia preparations, suggesting that PKCß mediated
odorant-stimulated phosphorylation (Bruch et al., 1997). These
data,
together with previous biochemical evidence (Boekhoff and Breer, 1992; Boekhoff et al., 1992), suggested that PKC phosphorylated
activated
7TMD receptors in olfactory neurons and played a role in receptor desensitization. Within this
context,
two 7TMD receptors known to be expressed in olfactory neurons—an odorant receptor (Ngai et al., 1993) and mGluR1 (Medler et al., 1998)—were used to test this hypothesis.
In this study we have shown that the mGluR1 PKC consensus sequence is phosphorylated by
both
PKCß and , although the extent of phosphorylation catalyzed by PKCß was
qualitatively
higher than that catalyzed by PKC. This apparent differential phosphorylation by the PKC
isotypes
may be indicative of their physiological roles in vivo. Activation of mGluR1 stimulates
phospholipase C and increases intracellular calcium (Pin and Duvoisin, 1995).
Increased intracellular
calcium may activate the calcium-sensitive PKCß that could then phosphorylate and
desensitize the
receptor. Since there are no known interactions between GRKs and mGluRs (Gereau
and
Heinemann, 1998), PKC may play a critical role in desensitization of these receptors.
The mGluR1 consensus phosphorylation sequence that we studied resides within the second
intracellular loop of the receptor, an important site for G-protein interaction in mGluRs (Francesconi and Duvoisin, 1998;Gereau and Heinemann,
1998).
Deletion of residues C694 and T695 within the second intracellular loop significantly reduced the
ability
of the receptor to couple to second messenger signaling pathways (Francesconi and
Duvoisin, 1998). Interestingly, T695 is the residue phosphorylated by PKCß and
in
our study. The mGluR1 second intracellular loop is also critical for activation of a phospholipase
C-coupled G-protein, and optimal coupling between the receptor and G-protein requires the 16
C-terminal residues of intracellular loop 2 (Gomeza et al., 1996). The
PKC consensus sequence tested in our study is also contained within the 16 C-terminal residues
of the
second intracellular loop. Taken with the results from other studies, the present study predicts
that the
PKC consensus sequence within intracellular loop 2 is a functional phosphorylation site that may
play a
role in receptor desensitization in vivo.
PKC, but not PKCß, phosphorylated the PKC consensus sequence within the
odorant
receptor. This was a surprising result since our previous study suggested that the ß isotype
probably mediated odorant-stimulated phosphorylation (Bruch et al., 1997). Since there are no additional PKC consensus sequences in the receptor, it is likely
that
PKCß interacts with other non-receptor components following receptor activation. Although
specific PKC substrates have not been identified in olfactory neurons, known substrates within
the
signaling pathway include phospholipase C, adenylate cyclase, IP3 receptor, GRKs
and
dynamin (Liu, 1996; Pronin and Benovic, 1997).
In this study we have described the identification of a novel extracellular phosphorylation
site in an
odorant receptor. Although this site was differentially phosphorylated by PKC in vitro,
the
possible functional significance of phosphorylation at this site in vivo is unknown.
Perhaps
PKC phosphorylation in the extracellular loop is associated with receptor synthesis, processing or
membrane targeting. Since these processes are not associated with acute increases in
intracellular
calcium, our observation of phosphorylation of the extracellular consensus site by the
calcium-insensitive PKC may have physiological relevance. Ongoing heterologous
expression
experiments will test this hypothesis.
Recent evidence suggests that the extracellular domains of 7TMD receptors may have
functional
significance beyond merely acting as static structural components (reviewed by Jiet al., 1998). For example, adrenergic receptor chimeras with mutations in the
third
extracellular loop exhibited altered affinity for agonist, presumably by affecting helical packing
within the
transmembrane domains (Zhao et al., 1998). A mechanism for
the
binding of thyrotropin releasing hormone to its receptor has also been described in which the
receptor
extracellular domains may act as primary binding sites that guide the hormone into the binding
site within
the membrane (Colson et al., 1998). A similar role for the
second
extracellular loop of the chemoattractant cAMP receptor of Dictyostelium has also been
proposed (Kim et al., 1997). It is of interest to note that the
second
extracellular loop of odorant receptors has also been proposed to function as a cell surface
identifier for
the particular receptor expressed in individual olfactory neurons (Singer et al., 1995). Although it is tempting to speculate that the odorant receptor
phosphorylation site in
the second extracellular loop may affect the conformation and ligand binding properties of the
receptor,
or may serve some role as a cell surface marker, additional experiments are required to determine
the
functional role of this novel PKC consensus sequence.
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Acknowledgments |
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This work was supported by NIH grant DC001500 (to R.C.B.). The authors thank H. Tran for excellent technical assistance.
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Accepted January 14, 1999
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