From the Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235
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ABSTRACT |
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FK506-binding protein (FKBP12) has been found to
be associated with the skeletal muscle ryanodine receptor (RyR1)
(calcium release channel), whereas FKBP12.6, a novel isoform of FKBP,
is selectively associated with the cardiac ryanodine receptor (RyR2). For both RyRs, the stoichiometry is 4 FKBP/RyR. Although FKBP12.6 differs from FKBP12 by only 18 of 108 amino acids, FKBP12.6 selectively binds to RyR2 and exchanges with bound FKBP12.6 of RyR2, whereas both
FKBP isoforms bind to RyR1 and exchange with bound FKBP12 of RyR1. To
assess the amino acid residues of FKBP12.6 that are critical for
selective binding to RyR2, the residues of FKBP12.6 that differ with
FKBP12 were mutated to the respective residues of FKBP12. RyR2 of
cardiac sarcoplasmic reticulum, prelabeled by exchange with
[35S]FKBP12.6, was used as assay system for
binding/exchange with the mutants. The triple mutant (Q31E/N32D/F59W)
of FKBP12.6 was found to lack selective binding to the cardiac RyR2,
comparable with that of FKBP12.0. In complementary studies, mutations
of FKBP12 to the three critical amino acids of FKBP12.6, conferred selective binding to RyR2. Each of the FKBP12.6 and FKBP12 mutants retained binding to the skeletal muscle RyR1. We conclude that three
amino acid residues (Gln31, Asn32, and
Phe59) of human FKBP12.6 account for the selective binding
to cardiac RyR2.
FK506, a fungal macrolide antibiotic, possesses potent
immunosuppressive activity. The drug has been used both experimentally and clinically to prevent organ graft rejection and to treat autoimmune diseases (1-5). FK506 suppresses activation of human T-lymphocytes by
blocking a key step required in the synthesis or activation of
transcription factors (NF-AT, AP-3, and NF- In skeletal muscle and heart, the ryanodine receptor (RyR)/calcium
release channel serves to release Ca2+ from sarcoplasmic
reticulum (SR), thereby triggering muscle contraction (10-12). FKBP12
has been found to be tightly associated with skeletal muscle RyR
(RyR1), and more recently its novel isoform, FKBP12.6, was found to be
selectively associated with cardiac RyR (RyR2). Thus, the structural
formulae for RyR1 and RyR2 are (RyR1
protomer)4(FKBP12)4 and (RyR2
protomer)4(FKBP12.6)4, respectively (13-16).
The ultrastructural localization of FKBP12 has been pinpointed within
the three dimensional structure of RyR1 by cryoelectron microscopy and
image enhancement analysis. There are four FKBPs/receptor with 4-fold
symmetry, and FKBP is located in the vicinity of RyR1 that is close to
the transverse tubule, consistent with its serving a role in E-C
coupling (17, 18). Both FKBP12 and FKBP12.6 share a number of
structural and functional similarities, including: 1) 85% amino acid
sequence homology (only 18 of 108 amino acid residues differ); 2)
PPIase activity that is inhibited by FK506 or rapamycin; and 3)
inhibition of calcineurin when the ternary complexes are formed with
FKBP and FK506 (6-8). FKBP12 appears to have a role in
excitation-contraction coupling in skeletal muscle by modulating the
release of Ca2+ from SR (15, 19, 20). However, the action
of FKBP12.6 on cardiac excitation-contraction coupling is less clear
(16). Recently, it has been suggested that FKBP12.6 may be involved in
the regulation of insulin biosynthesis and secretion by modulating intracellular Ca2+ levels via RyR2 in rat pancreatic
islets. It has been proposed that FKBP12.6 bound on RyR2 can be
released by cyclic ADP-ribose, a metabolite of NAD+ during
insulin secretion by glucose stimulation (21). The precise role of
FKBP12.6 in cardiac excitation-contraction coupling and insulin
secretion remains to be elucidated.
Although FKBP12.6 differs from FKBP12 by only 18 of 108 amino acid
residues2 (9, 35), FKBP12.6
selectively binds to cardiac RyR2 and exchanges with bound FKBP12.6 of
RyR2, whereas both FKBP isoforms bind to skeletal muscle RyR1 and
exchange with bound FKBP12 of RyR1 (16, 22). In the present study,
site-directed mutagenesis was used to determine the amino acid residues
of FKBP12.6 that confer its selective association with the cardiac RyR2.
Preparation of Human FKBP12.6 and FKBP12 Mutants--
The
preparation of human FKBP12.6 mutants was carried out using
site-directed mutagenesis system (QuickChangeTM
site-directed mutagenesis kit, Stratagene, La Jolla, CA). Briefly, the
complementary DNA coding region of human FKBP12.6 was subcloned into
the protein expression vector pET-3d at NcoI and
BamHI sites as described previously (9). Using this plasmid
as a DNA template, the mutants of FKBP12.6 were synthesized by
polymerase chain reaction in the presence of a pair of complementary
synthetic oligonucleotide primers containing the desired mutation using
Pfu DNA polymerase, which replicates both plasmid strands
with high fidelity and without displacing the mutant oligonucleotide
primers. Following temperature cycling of polymerase chain reaction,
the parental DNA template was digested by DpnI
endonuclease, which is specific for cleaving methylated and
hemimethylated DNA, and the nicked vector DNA incorporating the desired
mutations was then transformed into XL1-Blue supercompetent cells. The
mutants were identified by sequencing using an Applied Biosystems Prism
377 automatic sequencer (Applied Biosystems) with a Dye Terminator
Cycle Sequencing Ready Reaction kit (Perkin-Elmer). The mutants with
double or multiple amino acids mutations were subsequently made by
using the single or multiple mutants as a template. The
oligonucleotides primers used for mutagenesis were synthesized by
changing the DNA sequences of FKBP12.6 to FKBP12. The same procedure
described above was used to make FKBP12 mutants except using human
recombinant FKBP12, which was also subcloned in the pET-3d expression
vector, as a template and changing the DNA sequences from FKBP12 to
FKBP12.6.
Expression and Purification of FKBPs and Their Mutants--
The
pET-3d plasmid containing human FKBP12.6, FKBP12, and their mutants
were transformed into BL21 (DE3) competent cells, and the recombinant
and mutated proteins were induced by
isopropy- Expression and Purification of 35S-Labeled FKBP12.6
or 35S-Labeled FKBP12--
The biosynthesis of
35S-labeled FKBP12.6 or 35S-Labeled FKBP12 were
carried out essentially as described previously (9) with some
modification. Briefly, the pET-3d plasmid DNA containing human FKBP12.6
or FKBP12 was transformed into BL21 (DE3) host cell. Two ml of an
overnight culture from a single colony were inoculated into 200 ml of
M9 medium containing 50 µg/ml ampicillin and incubated at 37 °C
with shaking (250 rpm). The bacteria were collected by centrifugation
at 3000 rpm for 5 min (Beckman JA-14 rotor) when the OD600
value of the bacteria reached to 0.5. The pelleted bacteria were
resuspended in 200 ml of RPMI 1640 medium (Life Technologies, Inc.)
containing 50 mg/ml ampicillin, 1/40th of methionine, cystine, and
glucose concentration compared with the regular RPMI 1640 medium and
then isopropy- Isolation of Cardiac SR and Junctional Terminal Cisternae (TC) of
Skeletal Muscle--
Cardiac SR (CSR) was isolated from canine heart,
and TC vesicles of SR were isolated from fast twitch rabbit skeletal
muscle as described previously (24, 25). The R3 fraction
from the TC preparation of skeletal muscle SR was used in the
35S-labeled FKBP12 exchange experiments, because in
comparison with the highly enriched R4 TC fraction, which
has higher concentration of high affinity ryanodine binding (of about
25 pmol/mg protein), the concentration of high affinity ryanodine
binding site in the R3 fraction (about 8 pmol/mg of
protein) was in a similar range to that of the CSR (5 pmol/mg of protein).
Prelabeling of SR Vesicles by Exchange with
[35S]FKBP12.6 or [35S]FKBP12--
Cardiac
SR or skeletal muscle TC of SR (2.5 mg/ml) were prelabeled with
[35S]FKBP12.6 or [35S]FKBP12 (3 µM) by exchange as described previously by incubating 30 min at 37 °C (15, 16). The unbound [35S]FKBPs were
separated from the [35S]FKBPs bound on CSR or skeletal
muscle TC vesicles by sedimentation of 35,000 rpm (Beckman TL100.2
rotor) for 15 min at 4 °C. The cardiac SR or skeletal muscle TC
vesicles were resuspended in imidazole homogenization medium (5 mM imidazole-HCl, pH 7.4, and 0.3 M sucrose) to
a final concentration of 2.5 mg/ml.
Assay of Binding Affinity of FKBP and Mutants to Ryanodine
Receptors--
Skeletal muscle terminal cisternae of SR or cardiac SR
were prelabeled by exchange with [35S]FKBP12.6 or
[35S]FKBP12, respectively (see above), as described
previously (15, 16). The EC50 for the release of
[35S]FKBPs from such prelabeled SR by FKBPs and their
mutants was used an index of FKBP binding to the ryanodine receptors.
Briefly, [35S]FKBP12.6 and [35S]FKBP12 were
used to label cardiac SR and skeletal muscle TC, respectively. The SR
vesicles in a final concentration of 2 mg/ml were incubated with
increasing concentrations of FKBPs or mutants at 37 °C for 30 min.
The free [35S]FKBPs was separated from bound
[35S]FKBPs by diluting a 50 µl of sample (120 µg of
protein) into 200 µl of ice-cold imidazole homogenization medium
buffer and immediately sedimenting the vesicles by centrifugation
(95,000 rpm for 15 min at 4 °C). The pellet was quickly rinsed, then
resuspended in 200 µl of distilled water and counted in 5 ml of
Cytoscint liquid scintillation mixture.
Other Methods--
The protein concentration of SR membrane
fractions was determined by the Lowry procedure (26) and FKBPs and
mutants by scanning densitometry of Coomassie Blue-stained
SDS-polyacrylamide gel electrophoresis gels using a gel analysis and
image processing system (Technology Resources Inc., Nashville, TN)
(15). Bovine serum albumin was used as the protein standard.
SDS-polyacrylamide gel electrophoresis assay was performed with a
mini-slab gel apparatus (Hoeffer Scientific) using the buffer system
described by Laemmli (27).
Three Amino Acid Residues in FKBP12.6 Determine Its Selective
Binding to RyR2--
Human FKBP12.6 differs from FKBP12 by only 18 of
108 amino acids (9), yet FKBP12.6 selectively binds to and exchanges
with bound FKBP12.6 on cardiac RyR2, whereas both FKBP isoforms bind to
and exchange with bound FKBP12 of skeletal muscle RyR1 (16, 22). The
selective binding of FKBP12.6 to RyR2 must be referable to some of the
18 differing amino acid residues between the two FKBP isoforms (Fig.
1). In order to identify which amino acid residues determine selective binding and exchange, we mutated some of
the 18 differing amino acids of FKBP12.6 and assessed the decrease in
selective exchange to RyR2. Since binding and exchange have been found
to parallel one another, the EC50 for exchange,
i.e. the concentration of the FKBP and/or mutants to release
50% of [35S]FKBP12.6 served as an index of FKBP12.6
binding to RyR2. A total of 14 single or multiple recombinant mutants
of FKBP12.6 were made, and the concentration dependence for release of
[35S]FKBP12.6 from prelabeled heart SR was plotted (Fig.
2A and Table I). We first made seven single and one
double mutants by replacing the amino acids of FKBP12.6 with those of
FKBP12. In the calibration of the system, the EC50 for
exchange of FKBP12.6 to cardiac RyR2 was found to be approximately
620-fold lower than for FKBP12.0. That is, the EC50 of
FKBP12.6 for release of [35S]FKBP12.6 from cardiac SR by
exchange was 0.040 µM, compared with 24.9 µM for FKBP12. Two mutants of FKBP12.6, Q31E/N32D and F59W, with the EC50 values of 0.56 and 0.60 µM, respectively, have 14- and 15-fold decreased affinity
for cardiac RyR2, respectively, compared with FKBP12.6. Other single
mutants did not significantly decrease the affinity to cardiac RyR2.
That is, the EC50 values are in a similar range
(0.033-0.093 µM) (Table I and Fig. 2A) as
FKBP12.6 (0.040 µM). Based on these initial observations,
one double mutant and three triple mutants were made. The triple mutant (Q31E/N32D/F59W) was devoid of selective binding to cardiac SR having
an EC50 value of 19.7 µM, similar to FKBP12
(24.9 µM). Two other double mutants (Q31E/F59W and
N32D/F59W) were made to assess whether two amino acids from this triple
mutant were responsible for the selective binding to cardiac RyR2. The
results show that both double mutants (Q31E/F59W and N32D/F59W)
retained partial binding activity to cardiac RyR2 with EC50
values of 7.83 and 2.14 µM, respectively (Fig.
2A, Table I).
Mutations of FKBP12 Confer Selective Binding to RyR2--
If
mutation of these three amino acid residues in FKBP12.6 is sufficient
to wipe out selective binding to RyR2, then mutation of these residues
in FKBP12 to that of FKBP12.6 should confer selective binding. Three
mutants of FKBP12 (E31Q/D32N/W59F, E31Q/W59F, D32N/W59F) were made, in
which the three key amino acids of FKBP12 were replaced by the
respective residues of FKBP12.6. The results confirm that the triple
mutant (E31Q/D32N/W59F) binds to cardiac RyR2 with a similar
EC50 value (0.039 µM) as FKBP12.6 (0.040 µM). The double mutants (E31Q/W59F and D32N/W59F) bind
partially to cardiac RyR2, with EC50 values of 0.52 and
1.83 µM, respectively (Fig. 2B and Table
I).
Mutations in Key Amino Acids of FKBP12 and FKBP12.6 Do Not
Appreciably Modify Binding to RyR1--
A key control for validating
the role of the three amino acid residues to confer selective binding
to RyR2 is that the mutants retain their binding to RyR1. For this
propose, skeletal muscle SR prelabeled by exchange with
[35S]FKBP12 served as the assay system. We found that all
of the FKBP12.6 and FKBP12 mutants retained their ability to exchange with FKBP bound to skeletal muscle RyR1. That is, the mutants had
comparable binding affinity as FKBP12 or FKBP12.6, in the range of
0.037-0.3 µM (Fig. 3 and
Table II).
We conclude that three amino acids of FKBP12.6 (Gln31,
Asn32, and Phe59) determine selective binding
to RyR2.
In this study, insight is provided into the nature of the
interaction of FKBP with the RyRs of heart and skeletal muscle SR. FKBP12 and FKBP12.6 both bind to RyR1, whereas FKBP12.6 selectively binds to RyR2. In order to assess which amino acids of FKBP12.6 confer
selective binding to RyR2, we carried out site-directed mutagenesis in
nine amino acid residues that differ between the two FKBP isoforms. The
assay for selective binding/exchange of FKBP12.6 to RyR2 was then used
to assess which mutations alter the selectivity of binding.
We find that only three amino acid residues of FKBP12.6 determine the
selectivity of binding to RyR2. The triple amino acid mutation of
FKBP12.6 (Q31E/N32D/F59W), replacing the amino acids of FKBP12.6 with
the respective three amino acid residues in FKBP12, eliminates the
selective binding for RyR2. In complementary experiments, FKBP12 can be
made to selectively bind to RyR2 by mutating these same three amino
acid residues (E31Q/D32N/W59F) to those in FKBP12.6. The fact that all
mutants retain binding to skeletal muscle RyR1 with a similar affinity
as wild type FKBPs, provides evidence that the three-dimensional
structures of both FKBPs are stable and similar. We conclude that three
amino acid residues (Gln31, Asn32, and
Phe59) determine the selective binding of FKBP12.6 to the
cardiac RyR2.
The three-dimensional structures of FKBP12 and FKBP12-FK506 complexes
have been determined to high resolution by x-ray crystallography and
solution phase NMR (28-32). Since there is close sequence homology (87%) between FKBP12 and FKBP12.6 (9), we carried out structure modeling studies of FKBP12.6 and its mutants using the
three-dimensional structure of FKBP12 as the model. The 18 different
amino acid residues of FKBP12.6 were substituted, and energy
minimization was used to assess whether the three-dimensional structure
changes. Three-dimensional structure models of FKBP12.6 were generated using the SWISS-MODEL software system based on the three-dimensional structure of FKBP12 (Fig. 4). The model
of the structure of FKBP12.6 is quite similar to that of FKBP12. No
major conformational changes were indicated between wild type FKBP12.6
and its double and triple mutants (Q31E/N32D/F59W, Q31E/F59W, and
N32D/F59W) (not shown). Thus, no major structural changes are indicated
between the two FKBP isoforms or when mutating the three amino acid
residues that are critical for selective binding to RyR2.
INTRODUCTION
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ABSTRACT
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REFERENCES
B), which promote expression of lymphokine genes such as interleukin-2 (IL-2) (6). The
immunosuppressive action of the drug is related to its inhibition of
calcineurin, a calcium-dependent protein phosphatase
involved in intracellular signaling transduction, by way of binding to FK506-binding proteins
(FKBPs),1 a family of related
intracellular receptors including FKBP12 and FKBP12.6 (7-9).
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-D-thiogalactopyranoside. FKBPs and their
mutants were purified by high pressure liquid chromatography (HPLC)
using a TSK G3000SW column as described previously (23).
-D-thiogalactopyranoside (1 mM
final concentration) was added. A 2.5 mCi of
[35S]methionine and [35S]cystine mixture
(NEN Life Science Products) was then added to the medium after
incubation at 37 °C for 15 min with shaking, and the bacteria were
cultured for 5-6 h at the same conditions. The bacteria were collected
by centrifugation, and the pellets were resuspended in 10 ml of
phosphate-buffered saline and then lysed by sonication (50% pules,
30-s intervals for 3 min, Branson Sonic Power Co.). The supernatant was
collected by centrifugation at 15,000 × g for 30 min
at 4 °C, and the DNA was precipitated by adding protamine sulfate
(final concentration: 0.04%). Finally, the 35S-labeled
FKBP12.6 or 35S-labeled FKBP12 was purified by HPLC using a
TSK G3000SW column. The specific radioactivity of
[35S]FKBP12.6 or [35S]FKBP12 in the time
span of these experiments were changed from 6500 to 4500 cpm/pmol and
from 2100 to 1500 cpm/pmol, respectively.
RESULTS
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Fig. 1.
Comparison of the amino acid sequences of
human recombinant FKBP12 and FKBP12.6. The identical amino acids
are indicated by dots, and the different amino acids are
represented by single letter code for amino acid residues. Bold
letters indicate amino acids that have been mutated. The numbering
is shown on the top of the sequence. The amino acids that
are responsible for binding to cardiac ryanodine receptor (RyR2) in
FKBP12.6 are indicated with a star. Both FKBPs actually have
108 amino acids when counting the N-terminal methionine. We use the
numbering in the literature with the second amino acid (glycine) number
1 in order to be consistent with the literature.
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Fig. 2.
Exchange of FKBPs mutants with
[35S]FKBP12.6 prelabeled CSR. CSR vesicles (2.5 mg/ml, Ref. 24) were prelabeled by exchange with
[35S]FKBP12.6 (3 µM) by incubation for 30 min at 37 °C. The displacement of [35S]FKBP12.6 from
prelabeled CSR was carried out at 37 °C for 30 min in the presence
of varying concentrations of FKBP12, FKBP12.6, and FKBP12.6 mutants
(A) or FKBP12 mutants (B). The
[35S]FKBP12.6 remaining in the CSR vesicles was
determined as described under "Experimental Procedure." Each
experiment was performed with two or three different preparations with
each point in duplicate. The EC50 values are tabulated in
Table I.
Comparison of the EC50 and the relative exchange efficiency
(REE) of FKBP12, FKBP12.6, and their mutants on
[35S]FKBP12.6-labeled CSR
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Fig. 3.
Exchange of FKBP mutants with
[35S]FKBP12 prelabeled skeletal muscle terminal
cisternae. Skeletal muscle terminal cisternae of sarcoplasmic
reticulum (R3 fraction, 2.5 mg/ml, Ref. 25) were
prelabeling by exchange of skeletal muscle TC (R3 fraction)
vesicles with [35S]FKBP12 (3 µM). The
displacement of [35S]FKBP12 from prelabeled skeletal
muscle TC by FKBPs mutants was carried out as described in Fig. 2. Each
experiment was performed with two or three different preparations with
each point in duplicate. The EC50 values are tableted in
Table II.
Comparison of the EC50 and the relative exchange efficiency
(REE) of FKBP12, FKBP12.6, and their mutants on
[35S]FKBP12-labeled TC of skeletal muscle SR
DISCUSSION
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ABSTRACT
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Fig. 4.
The three-dimensional structure of
FKBP12.6. The structure of FKBP12.6 was generated using
SWISS-MODEL software system based on the three-dimensional structure of
FKBP12. The FK506 binds in the hydrophobic pocket contoured by
Tyr26, Phe46, Trp59,
Tyr82, and Phe99. The three amino acid residues
that determine the specificity of binding to RyR2 are indicated by
their space filling side chains (Asn31, Gln32,
and Phe59). The numbering of all of these amino acid
residues would be increased by one, were the N-terminal residue
(methionine) numbered 1 instead of 0.2
Schreiber and his colleagues reported that Tyr26, Phe46, Phe48, Trp59, Tyr82, and Phe99 of FKBP12 pack together to form a well defined hydrophobic core where FK506 and rapamycin bind. The indole ring of Trp59 is at the end of the pocket and serves as a platform for the pipecolinyl ring of FK506 (29, 30). It is likely that there is a similar hydrophobic pocket in FKBP12.6 for binding to FK506 as for FKBP12. The amino acids that form the pocket are conserved in both FKBPs with the exception of Phe59 in FKBP12.6, which replaces Trp59 in FKBP12. The hydrophobic characteristics of the pocket in FKBP12.6 would not be expected to be changed very much when Phe59 is mutated to Trp59, since both are hydrophobic amino acids. However, these two hydrophobic amino acid residues differ somewhat in size, so that steric constraints may contribute in part to the stability of interaction. The pocket is somewhat larger in FKBP12.6, since it contains the smaller phenylalanine residue instead of the tryptophan residue in FKBP12. Schreiber and co-workers (33) studied the substrate specificity of rotamase enzymic activity of FKBP12 and suggested that FK506 acts as a peptidomimetic of leucine (twisted amide)-proline dipeptide. Snyder and co-workers (34) further suggested that FKBP12 binds RyR1 at a conserved valine-proline peptide sequence (LSRLVPLDDLV). There is a similar conserved proline in cardiac RyR2 (LRSLIPLDDLV) but containing an isoleucine instead of a valine. The larger isoleucine in such a binding domain of RyR2 may be accommodated better by the larger pocket of FKBP12.6, which contains the smaller phenylalanine instead of tryptophan. Thus, steric factors may in part determine the selective binding of FKBP12.6 to cardiac RyR2, whereas both FKBPs bind to skeletal muscle RyR1.
The decreased affinity for binding to cardiac RyR2 by mutating Gln31 and Asn32 of FKBP12.6 to Glu31 and Asp32 of FKBP12 represents an increase in two negative charges. This would suggest that charge repulsion may be a factor in the lack of binding of FKBP12 to RyR2.
Our studies pinpoint the amino acid residues of FKBP12.6 that determine
the selective interaction with RyR2. Since the structure of FKBP12.6
and FKBP12 appear to be similar, we infer that a difference in the
structure of RyR1 and RyR2 also contributes to the selectivity of the interaction.
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ACKNOWLEDGEMENTS |
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We thank Dr. Gregory Wiederrecht, Department of Immunology, Merck Research Laboratories, for providing us human FKBP12 and FKBP12.6 plasmids. We also thank Dr. Andre Krezel, Department of Molecular Biology, Vanderbilt University, for helping us with the computer modeling of FKBP12.6 (Fig. 4).
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant HL32711 and the Muscular Dystrophy Association (to S. F.).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.
To whom correspondence should be addressed: Dept. of Molecular
Biology, Vanderbilt University, Nashville, TN 37235. Tel.: 615-322-2132; Fax: 615-343-6833; E-mail:
sidney.fleischer{at}vanderbilt.edu.
2 We are using the numbering system in the literature (35) for consistency (see Figs. 1 and 4), that is, the N-terminal methionine of FKBP12 is designated as the 0 amino acid residue so that the C-terminal glycine is numbered 107.
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ABBREVIATIONS |
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The abbreviations used are: FKBP, FK506-binding protein; CSR, cardiac SR; FKBP12, a 12-kDa FKBP; FKBP12.6, an isoform of FKBP with slightly slower mobility than FKBP12; RyR1 and RyR2, ryanodine receptor isoform 1 from skeletal muscle and isoform 2 from heart, respectively; SR, sarcoplasmic reticulum; TC, terminal cisternae of SR; HPLC, high performance liquid chromatography.
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