J Biol Chem. 2004 Dec 17;279(51):53674-82. Epub 2004 Oct 7.
Interaction between protein kinase Cmu and the vanilloid receptor type 1.
Wang, Y., Kedei, N., Wang, M., Wang, Q. J., Huppler, A. R., Toth, A., Tran, R., Blumberg, P. M.,
["NCI, National Institutes of Health, Bethesda, MD 20892-4255, USA. wangy66@bjmu.edu.cn"]
["NCI, National Institutes of Health, Bethesda, MD 20892-4255, USA. wangy66@bjmu.edu.cn"]
The capsaicin receptor VR1 is a polymodal nociceptor activated by multiple stimuli. It has been reported that protein kinase C plays a role in the sensitization of VR1. Protein kinase D/PKCmu is a member of the protein kinase D serine/threonine kinase family that exhibits structural, enzymological, and regulatory features distinct from those of the PKCs, with which they are related. As part of our effort to optimize conditions for evaluating VR1 pharmacology, we found that treatment of Chinese hamster ovary (CHO) cells heterologously expressing rat VR1 (CHO/rVR1) with butyrate enhanced rVR1 expression and activity. The expression of PKCmu and PKCbeta1, but not of other PKC isoforms, was also enhanced by butyrate treatment, suggesting the possibility that these two isoforms might contribute to the enhanced activity of rVR1. In support of this hypothesis, we found the following. 1) Overexpression of PKCmu enhanced the response of rVR1 to capsaicin and low pH, and expression of a dominant negative variant of PKCmu reduced the response of rVR1. 2) Reduction of endogenous PKCmu using antisense oligonucleotides decreased the response of exogenous rVR1 expressed in CHO cells as well as of endogenous rVR1 in dorsal root ganglion neurons. 3) PKCmu localized to the plasma membrane when overexpressed in CHO/rVR1 cells. 4) PKCmu directly bound to rVR1 expressed in CHO cells as well as to endogenous rVR1 in dorsal root ganglia or to an N-terminal fragment of rVR1, indicating a direct interaction between PKCmu and rVR1. 5) PKCmu directly phosphorylated rVR1 or a longer N-terminal fragment (amino acids 1-118) of rVR1 but not a shorter one (amino acids 1-99). 6) Mutation of S116A in rVR1 blocked both the phosphorylation of rVR1 by PKCmu and the enhancement by PKCmu of the rVR1 response to capsaicin. We conclude that PKCmu functions as a direct modulator of rVR1.
PMID: 15471852

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Screening
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Experimental screening | Non-experimental screening | Reference | ||||||||
TRP channel construct | Interactor source | |||||||||
TRP channel | Interactor | Method | Species | Region | Species | Organ/tissue | Sample type | |||
TRPV1 |
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PRKD1/PKCƬ | Inference | Prediction | 15471852 |
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Validation: In vitro validation
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Assay with recombinant proteins | Reference | |||||||||
TRP channel construct | Interactor construct | |||||||||
TRP channel | Interactor | Method | Species | Region | Expression system | Species | Region | Expression system | ||
TRPV1 |
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PRKD1/PKCƬ | Fusion protein-pull down assay | Rat | Full-length | E. coli | Not specified | Full-length | Not specified | 15471852 |
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Validation: In vivo validation
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Assay with endogenous proteins | Assay with overexpressed proteins | Reference | ||||||||
Cell or tissue | Cell or tissue | TRP channel construct | Interactor construct | |||||||
TRP channel | Interactor | Method | Species | Region | Species | Region | ||||
TRPV1 |
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PRKD1/PKCƬ | Co-immunoprecipitation | CHO | Rat | Full-length | Human | Full-length | 15471852 | |
TRPV1 |
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PRKD1/PKCƬ | Co-immunoprecipitation | Rat dorsal root ganglion neuron | 15471852 |
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Characterization
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Binding region mapping | Stoichiometry | Affinity (Kd) | Reference | |||||||
TRP channel | Interactor | |||||||||
TRP channel | Interactor | Method | Species | Region | Species | Region | ||||
TRPV1 |
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PRKD1/PKCƬ | Fusion protein-pull down assay | Rat | 99-118 | Human | Not determined | 15471852 |
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Functional consequence
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TRP channel | Interactor | Method | Post-translational modification | Subcellular trafficking | Activity | Reference | ||||||
TRPV1 |
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PRKD1/PKCƬ | In vitro PTM assay | Phosphorylation (Ser-116) | 15471852 | |||||||
TRPV1 |
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PRKD1/PKCƬ | Calcium measurement | Activation | 15471852 |
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