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.External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c

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"]
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: 15471852External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c
Screening Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Experimental screening Non-experimental screening Reference
TRP channel construct Interactor source
TRP channel Interactor Method Species Region Species Organ/tissue Sample type
TRPV1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PRKD1/PKCƬ Inference Prediction 15471852
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Validation: In vitro validation Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Assay with recombinant proteins Reference
TRP channel construct Interactor construct
TRP channel Interactor Method Species Region Expression system Species Region Expression system
TRPV1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PRKD1/PKCƬ Fusion protein-pull down assay Rat Full-length E. coli Not specified Full-length Not specified 15471852
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Validation: In vivo validation Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  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 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PRKD1/PKCƬ Co-immunoprecipitation CHO Rat Full-length Human Full-length 15471852
TRPV1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PRKD1/PKCƬ Co-immunoprecipitation Rat dorsal root ganglion neuron 15471852
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Characterization Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Binding region mapping Stoichiometry Affinity (Kd) Reference
TRP channel Interactor
TRP channel Interactor Method Species Region Species Region
TRPV1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PRKD1/PKCƬ Fusion protein-pull down assay Rat 99-118 Human Not determined 15471852
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Functional consequence Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
TRP channel Interactor Method Post-translational modification Subcellular trafficking Activity Reference
TRPV1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PRKD1/PKCƬ In vitro PTM assay Phosphorylation (Ser-116) 15471852
TRPV1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PRKD1/PKCƬ Calcium measurement Activation 15471852
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
TRP / Interactor

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