J Biol Chem. 2008 Nov 21;283(47):32821-30. doi: 10.1074/jbc.M805382200. Epub 2008 Aug 28.

TRPC3 controls agonist-stimulated intracellular Ca2+ release by mediating the interaction between inositol 1,4,5-trisphosphate receptor and RACK1.External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c

Bandyopadhyay, B. C., Ong, H. L., Lockwich, T. P., Liu, X., Paria, B. C., Singh, B. B., Ambudkar, I. S.,
["Secretory Physiology Section, Molecular Physiology and Therapeutics Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892, USA."]
Activation of TRPC3 channels is concurrent with inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R)-mediated intracellular Ca(2+) release and associated with phosphatidylinositol 4,5-bisphosphate hydrolysis and recruitment to the plasma membrane. Here we report that interaction of TRPC3 with receptor for activated C-kinase-1 (RACK1) not only determines plasma membrane localization of the channel but also the interaction of IP(3)R with RACK1 and IP(3)-dependent intracellular Ca(2+) release. We show that TRPC3 interacts with RACK1 via N-terminal residues Glu-232, Asp-233, Glu-240, and Glu-244. Carbachol (CCh) stimulation of HEK293 cells expressing wild type TRPC3 induced recruitment of a ternary TRPC3-RACK1-IP(3)R complex and increased surface expression of TRPC3 and Ca(2+) entry. Mutation of the putative RACK1 binding sequence in TRPC3 disrupted plasma membrane localization of the channel. CCh-stimulated recruitment of TRPC3-RACK1-IP(3)R complex as well as increased surface expression of TRPC3 and receptor-operated Ca(2+) entry were also attenuated. Importantly, CCh-induced intracellular Ca(2+) release was significantly reduced as was RACK1-IP(3)R association without any change in thapsigargin-stimulated Ca(2+) release and entry. Knockdown of endogenous TRPC3 also decreased RACK1-IP(3)R association and decreased CCh-stimulated Ca(2+) entry. Furthermore, an oscillatory pattern of CCh-stimulated intracellular Ca(2+) release was seen in these cells compared with the more sustained pattern seen in control cells. Similar oscillatory pattern of Ca(2+) release was seen after CCh stimulation of cells expressing the TRPC3 mutant. Together these data demonstrate a novel role for TRPC3 in regulation of IP(3)R function. We suggest TRPC3 controls agonist-stimulated intracellular Ca(2+) release by mediating interaction between IP(3)R and RACK1.
PMID: 18755685External 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
TRPC3 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 RACK1 Yeast two-hybrid Not specified 1-341 Human Embroynic kidney cDNA library 18755685
(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
TRPC3 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 RACK1 Co-immunoprecipitation HEK293 Not specified Full-length Not used 18755685
TRPC3 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 RACK1 Co-immunoprecipitation Rat brain lysates 18755685
TRPC3 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 RACK1 Co-immunofluorescence staining HEK293 Not specified Full-length Not used 18755685
(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
TRPC3 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 RACK1 Yeast two-hybrid Not specified 221-342 Human Not determined 18755685
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
TRP / Interactor

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