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J. Biol. Chem., Vol. 276, Issue 42, 38762-38773, October 19, 2001
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From the Rammelkamp Center for Education and Research, MetroHealth
Medical Center, and Department of Physiology and Biophysics, Case
Western Reserve University School of Medicine,
Cleveland, Ohio 44106
Transient receptor
potential and transient receptor potential-like (TRPL) are
Ca2+-permeable cation channels found in
Drosophila photoreceptor cells associated with large
multimeric signaling complexes held together by the scaffolding
protein, INAD. To identify novel proteins involved in channel
regulation, Drosophila INAD was used as bait in a yeast two-hybrid screen of a Drosophila head cDNA library.
Sequence analysis of one identified clone showed it to be identical to the Drosophila homolog of human FK506-binding protein,
FKBP52 (previously known as FKBP59). To determine the function of
dFKBP59, TRPL channels and dFKBP59 were co-expressed in Sf9
cells. Expression of dFKBP59 produced an inhibition of Ca2+
influx via TRPL in fura-2 assays. Likewise, purified recombinant dFKBP59 produced a graded inhibition of TRPL single channel activity in
excised inside-out patches when added to the cytoplasmic membrane surface. Immunoprecipitations from Sf9 cell lysates using
recombinant tagged dFKBP59 and TRPL showed that these proteins directly
interact with each other and with INAD. Addition of FK506 prior to
immunoprecipitation resulted in a temperature-dependent
dissociation of dFKBP59 and TRPL. Immunoprecipitations from
Drosophila S2 cells and from fly head lysates demonstrated
that dFKBP59, but not dFKBP12, interacts with TRPL in vivo.
Likewise, INAD immunoprecipitates with dFKBP59 from S2 cell and head
lysates. Immunocytochemical evaluation of thin sections of fly heads
revealed specific FKBP immunoreactivity associated with the eye.
Site-directed mutagenesis showed that mutations of P702Q or
P709Q in the highly conserved TRPL sequence 701LPPPFNVLP709 eliminated interaction of the
TRPL with dFKBP59. These results provide strong support for the
hypothesis that immunophilin dFKBP59 is part of the TRPL-INAD signaling
complex and plays an important role in modulation of channel activity
via interaction with conserved leucyl-prolyl dipeptides located near
the cytoplasmic mouth of the channel.
Regulation of Drosophila TRPL Channels by
Immunophilin FKBP59*
,
*
This work was supported in part by NIGMS Grant GM52019 from
the National Institutes of Health (to W. P. S.) and Postdoctoral Fellowship Award 20381B from the American Heart Association, Northeast Ohio Affiliate (to M. G.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Current address: Cancer Research Campaign Beatson Laboratories
Bearsden, Glasgow G61 1BD, UK.
§
To whom correspondence should be addressed: Rammelkamp Center, Rm.
R322, MetroHealth Medical Center, 2500 MetroHealth Dr., Cleveland, OH
44109-1998. Tel.: 216-778-8965; Fax: 216-778-8997; E-mail:
wschilling@metrohealth.org.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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