Biological constraints limit the use of rapamycin-inducible FKBP12-Inp54p for depleting PIP2 in dorsal root ganglia neurons

Jaeda C Coutinho-Budd,Samuel B Snider,Mark J Zylka,Joseph E Rittiner,Brendan J Fitzpatrick

doi:10.1186/1477-5751-12-13

Abstract

BackgroundRapamycin-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.ResultsWe 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 (CGRPα) 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.ConclusionsTaken 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.

Highlights

Rapamycin-induced translocation systems can be used to manipulate biological processes with precise temporal control
Rapamycin induces translocation of Venus-FK506 Binding Protein 12 (FKBP12)-Inp54p from the cytoplasm to plasma membrane in cell lines Before generating knockin mice, we set out to verify that the rapamycin-inducible phosphatase components functioned in our hands as described [3,4]
Rapamycin treatment did not affect behavior in Rosa-FKBP Rapamycin Binding (FRB) PLF/CGRP-Inp54p heterozygous mice in vivo We recently found that PIP2 levels in dorsal root ganglia (DRG) at the time of inflammation enduringly affected thermal hypersensitivity, with preemptively reduced PIP2 levels correlating with an enduring reduction in thermal hypersensitivity [22]

Summary

Introduction

Rapamycin-induced translocation systems can be used to manipulate biological processes with precise temporal control. The immunosuppressant macrolide, rapamycin, induces the dimerization of two naturally occurring protein domains: FK506 Binding Protein 12 (FKBP12) with the FKBP Rapamycin Binding (FRB) domain of mTOR [1] These domains can be attached to other proteins to temporally and spatially control cell signaling with rapamycin or rapamycin analogs. Two groups used these domains to directly and selectively deplete the lipid PIP2 in cultured cells [3,4] and show that PIP2 was important for GPCR signaling and ion channel function [7,8,9] Both groups used 1) a plasma membrane-anchored FRB domain and 2) a cytosolic PIP2-specific phosphatase (yeast Inositol polyphosphate 5-phophatase (Inp54p) or mammalian type IV 5-phosphatase) fused to FKBP12.

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