PMID- 24010830
OWN - NLM
STAT- MEDLINE
DCOM- 20140710
LR  - 20211021
IS  - 1477-5751 (Electronic)
IS  - 1477-5751 (Linking)
VI  - 12
DP  - 2013 Sep 8
TI  - Biological constraints limit the use of rapamycin-inducible FKBP12-Inp54p for 
      depleting PIP2 in dorsal root ganglia neurons.
PG  - 13
LID - 10.1186/1477-5751-12-13 [doi]
AB  - BACKGROUND: Rapamycin-induced translocation systems can be used to manipulate 
      biological processes with precise temporal control. These systems are based on 
      rapamycin-induced dimerization of FK506 Binding Protein 12 (FKBP12) with the FKBP 
      Rapamycin Binding (FRB) domain of mammalian target of rapamycin (mTOR). Here, we 
      sought to adapt a rapamycin-inducible phosphatidylinositol 4,5-bisphosphate 
      (PIP2)-specific phosphatase (Inp54p) system to deplete PIP2 in nociceptive dorsal 
      root ganglia (DRG) neurons. RESULTS: We genetically targeted membrane-tethered 
      CFP-FRBPLF (a destabilized FRB mutant) to the ubiquitously expressed Rosa26 
      locus, generating a Rosa26-FRBPLF knockin mouse. In a second knockin mouse line, 
      we targeted Venus-FKBP12-Inp54p to the Calcitonin gene-related peptide-alpha 
      (CGRPalpha) locus. We hypothesized that after intercrossing these mice, rapamycin 
      treatment would induce translocation of Venus-FKBP12-Inp54p to the plasma 
      membrane in CGRP+ DRG neurons. In control experiments with cell lines, rapamycin 
      induced translocation of Venus-FKBP12-Inp54p to the plasma membrane, and 
      subsequent depletion of PIP2, as measured with a PIP2 biosensor. However, 
      rapamycin did not induce translocation of Venus-FKBP12-Inp54p to the plasma 
      membrane in FRBPLF-expressing DRG neurons (in vitro or in vivo). Moreover, 
      rapamycin treatment did not alter PIP2-dependent thermosensation in vivo. 
      Instead, rapamycin treatment stabilized FRBPLF in cultured DRG neurons, 
      suggesting that rapamycin promoted dimerization of FRBPLF with endogenous FKBP12. 
      CONCLUSIONS: Taken together, our data indicate that these knockin mice cannot be 
      used to inducibly deplete PIP2 in DRG neurons. Moreover, our data suggest that 
      high levels of endogenous FKBP12 could compete for binding to FRBPLF, hence 
      limiting the use of rapamycin-inducible systems to cells with low levels of 
      endogenous FKBP12.
FAU - Coutinho-Budd, Jaeda C
AU  - Coutinho-Budd JC
AD  - Curriculum in Neurobiology, University of North Carolina at Chapel Hill, Chapel 
      Hill, NC 27599, USA. zylka@med.unc.edu.
FAU - Snider, Samuel B
AU  - Snider SB
FAU - Fitzpatrick, Brendan J
AU  - Fitzpatrick BJ
FAU - Rittiner, Joseph E
AU  - Rittiner JE
FAU - Zylka, Mark J
AU  - Zylka MJ
LA  - eng
GR  - P30 NS045892/NS/NINDS NIH HHS/United States
GR  - R01 NS067688/NS/NINDS NIH HHS/United States
GR  - 1F31NS068038/NS/NINDS NIH HHS/United States
GR  - P30NS045892/NS/NINDS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20130908
PL  - England
TA  - J Negat Results Biomed
JT  - Journal of negative results in biomedicine
JID - 101152210
RN  - 0 (Inositol Phosphates)
RN  - 0 (Peptides)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 93060-87-8 (inositol 4,5-bisphosphate)
RN  - EC 3.1.3.16 (Phosphoprotein Phosphatases)
RN  - EC 5.2.1.- (Tacrolimus Binding Protein 1A)
RN  - JHB2QIZ69Z (Calcitonin Gene-Related Peptide)
RN  - W36ZG6FT64 (Sirolimus)
SB  - IM
MH  - Animals
MH  - Biosensing Techniques
MH  - Calcitonin Gene-Related Peptide/metabolism
MH  - Cell Membrane/drug effects/metabolism
MH  - Cells, Cultured
MH  - Ganglia, Spinal/drug effects/*metabolism
MH  - HEK293 Cells
MH  - Heterozygote
MH  - Humans
MH  - Hypersensitivity/pathology
MH  - Inflammation/pathology
MH  - Inositol Phosphates/*metabolism
MH  - Mice
MH  - Models, Biological
MH  - Neurons/drug effects/*metabolism
MH  - Peptides/metabolism
MH  - Phosphoprotein Phosphatases/*metabolism
MH  - Protein Transport/drug effects
MH  - Rats
MH  - Recombinant Fusion Proteins/*metabolism
MH  - Sensory Receptor Cells/drug effects/metabolism
MH  - Sirolimus/*pharmacology
MH  - Tacrolimus Binding Protein 1A/*metabolism
PMC - PMC3844522
EDAT- 2013/09/10 06:00
MHDA- 2014/07/11 06:00
PMCR- 2013/09/08
CRDT- 2013/09/10 06:00
PHST- 2013/05/30 00:00 [received]
PHST- 2013/09/05 00:00 [accepted]
PHST- 2013/09/10 06:00 [entrez]
PHST- 2013/09/10 06:00 [pubmed]
PHST- 2014/07/11 06:00 [medline]
PHST- 2013/09/08 00:00 [pmc-release]
AID - 1477-5751-12-13 [pii]
AID - 10.1186/1477-5751-12-13 [doi]
PST - epublish
SO  - J Negat Results Biomed. 2013 Sep 8;12:13. doi: 10.1186/1477-5751-12-13.