(Received for publication, May 16, 1995; and in revised form, August 11, 1995)
From the
FK506, an immunosuppressant that prolongs allograft survival, is
a co-drug with its intracellular receptor, FKBP12. The
FKBP12FK506 complex inhibits calcineurin, a critical signaling
molecule during T-cell activation. FKBP12 was, until recently, the sole
FKBP known to mediate calcineurin inhibition at clinically relevant
FK506 concentrations. The best characterized cellular function of
FKBP12 is the modulation of ryanodine receptor isoform-1, a component
of the calcium release channel of skeletal muscle sarcoplasmic
reticulum.
Recently, a novel protein, FKBP12.6, was found to inhibit calcineurin at clinically relevant FK506 concentrations. We have cloned the cDNA encoding human FKBP12.6 and characterized the protein. In transfected Jurkat cells, FKBP12.6 is equivalent to FKBP12 at mediating the inhibitory effects of FK506. Upon binding rapamycin, FKBP12.6 complexes with the 288-kDa mammalian target of rapamycin. In contrast to FKBP12, FKBP12.6 is not associated with ryanodine receptor isoform-1 but with the distinct ryanodine receptor isoform-2 in cardiac muscle sarcoplasmic reticulum. Our results suggest that FKBP12.6 has both a unique physiological role in excitation-contraction coupling in cardiac muscle and the potential to contribute to the immunosuppressive and toxic effects of FK506 and rapamycin.
FK506 (tacrolimus) is a powerful immunosuppressive drug for
treating graft rejection and autoimmune disorders. Rapamycin (RAP, ()sirolimus) is an immunosuppressant structurally-related to
FK506 but with a distinct mechanism of action. Both drugs bind to a
family of intracellular receptors, the FK506 binding proteins (FKBPs),
whose members include FKBPs 12, 12.6, 13, 25, 51, and 52 (for review,
see (1) ). All FKBPs are peptidyl-prolyl isomerases, catalyzing
the cis-trans isomerization of peptidyl-prolyl bonds in
peptides and proteins, an activity inhibited by both FK506 and RAP.
Peptidyl-prolyl isomerase inhibition is unrelated to
immunosuppression. FK506 and RAP gain function upon binding FKBP12. The
FKBP12FK506 and FKBP12
RAP complexes are the actual
immunosuppressive species whose targets are calcineurin (CaN) and the
mammalian target of RAP (mTOR), respectively (for review, see (1) and (2) ). CaN is a Ca
-dependent,
serine-threonine phosphatase required during the commitment phase
(G
G
) of T-cell activation(3) .
Inhibition of CaN blocks the nuclear translocation of transcription
factors such as nuclear factor of activated T-cells and NF-
B,
controlling the expression of cytokine genes whose products are
required for immune response coordination (for review, see (2) ). RAP, unlike FK506, does not block lymphokine production
but inhibits the T-cell proliferative response to cytokines by blocking
G
S-phase progression. The function of mTOR, a
288-kDa protein related to phosphatidylinositol kinases, is unknown.
CaN is a ubiquitous protein, and its inhibition at unwanted sites is
most responsible for the toxicity associated with FK506
therapy(4) . That immunosuppression and toxicity are
mechanistically linked through inhibition of CaN has been documented
using the nonimmunosuppressive and nontoxic FK506 analog, L-685,818
(`818; see Fig. 1). The observations that `818 binds tightly to
FKBP12, that the human FKBP12`818 complex has little affinity for
CaN, and that `818 reverses FK506 toxicity have demonstrated that CaN
inhibition, not FKBP binding, is responsible for the toxicity profile
of FK506(4, 5) .
Figure 1: Structures of the FKBP12.6 and FKBP12 ligands.
The cellular and pharmacologic
functions of FKBP12 are unrelated. Physiologically, FKBP12 regulates
the ryanodine receptor (RyR-1), an intracellular
Ca-release channel (CRC) required for
excitation-contraction coupling in skeletal muscle. The native CRC,
isolated from the terminal cisternae (TC) of skeletal muscle
sarcoplasmic reticulum (SR), is composed of four 565-kDa RyR-1
protomers and four FKBP12 molecules(6) . FKBP-stripped CRC
differs functionally from normal channels. It is activated by lower
concentrations of caffeine (6, 7) or
Ca
(8, 9) , higher Mg
concentrations are required for inactivation(9) , and it
has a greater open probability and displays longer mean open times in
the full conductance state(8) . These effects, reversed upon
rebinding FKBP12, indicate that FKBP12 stabilizes a closed conformation
of the channel. Cloned RyR-1, expressed in insect cells, also exhibits
channel properties functionally different from those of the native
CRC(7) . Without FKBP12, the channel flickers among
subconductance states, while co-expression of FKBP12 and RyR-1
generates channels opening to the full-conductance state(7) ,
suggesting that FKBP12 insures cooperativity among RyR-1 protomers.
FKBP12 may asymmetrically regulate ion flow through the channel,
promoting the flow of Ca
unidirectionally from the
lumen of the SR to the cytoplasm during channel
activation(10) .
Until recently, FKBP12 was the sole FKBP believed to be relevant to FK506-mediated immunosuppression or toxicity. It had been the only FKBP known to be a potent mediator of FK506's inhibition of CaN in vitro(11) and signal transduction in Jurkat cells(12) . Recently, a novel FKBP, FKBP12.6, was purified, sequenced, and characterized biochemically(13) . Closely related to FKBP12, it has the same number of amino acids and 18 mostly conservative amino acid substitutions (13) . The most striking substitution is that of a phenylalanine for a highly conserved tryptophan (13) forming the base of the drug-binding cavity. FKBP12.6 and FKBP12 have equal affinities for FK506 and are equipotent mediators of CaN inhibition by FK506(13) . Thus, FKBP12.6 has the potential to mediate the immunosuppression or toxicity associated with FK506 therapy.
We have cloned and expressed the cDNA encoding human FKBP12.6. The characterization of human FKBP12.6 in the presence and absence of its drug ligands is the subject of this report.
The secondary sense primers, corresponding to amino acids 14-19, RTFPKK, were (A/C)GNACNTTYCCNAAGAA and (A/C)GNACNTTYCCNAAAAA. The secondary antisense primers, corresponding to amino acids 60-65, FEEGAA, were GCNGCNCCYTCYTCGA and GCNGCNCCYTCYTCAA. One µl of a 1:10 dilution of each primary PCR reaction mixture was used in each of the four possible secondary PCR reactions (48 reactions total). The combination of the (A/C)GNACNTTYCCNAAGAA and GCNGCNCCYTCYTCAA primers gave the greatest amount of 156-base pair product. These two primers, resynthesized with EcoRI linkers attached, were used to generate a product that was digested with EcoRI, subcloned into the EcoRI site of pUC19, and sequenced to confirm that it encoded a fragment of hFKBP12.6.
The remainder of the cDNA encoding hFKBP12.6 was cloned using the rapid amplification of cDNA ends) (RACE) technique. To obtain the 3` end of the cDNA, a sense primer, RCCTTTCAAGTTCAGAA, corresponding to a specific sequence obtained from the partial hFKBP12.6 cDNA clone obtained above, was used. The first antisense primer, GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT, anneals to poly(A) tracts. The PCR reactions were performed as described previously (14) , and 1 µl of the 10-fold diluted product was used in a secondary PCR reaction. In the second PCR reaction, the sense primer (corresponding to a specific nucleotide sequence in the original partial cDNA) was AAACAGGAAGTCATCAA, and the antisense primer was GACTCGAGTCGACATCG. The PCR reactions were performed as described for the primary amplification; the products were purified by agarose gel electrophoresis, and the major 800-base pair product was reamplified with the same set of secondary primers except that the sense primer contained an EcoRI linker. The product was cloned between the EcoRI and SalI sites of pUC19.
To obtain the 5` end of the gene, human brain 5`-RACE-Ready cDNA (Clontech) was used as the template. A 5` anchor primer was supplied by the manufacturer. The first antisense primer (corresponding to a nucleotide sequence in the original partial cDNA) used was TTGATGACTTCCTGTTTGCCAATTC. The PCR conditions were as described for the 3` RACE reactions. The products of the first reaction were diluted 10-fold, and those used in a second PCR reaction with the manufacturer's anchor primer and a second antisense primer, GAAAGGYTTGTTTCTGTCTCTGGAT. The product of the secondary reaction was reamplified using an EcoRI-linkered antisense primer, and the product was subcloned into the EcoRI site of pUC19 (the Race-Ready cDNA contains an EcoRI site at the 5` end) and sequenced. Alignment of the 5` RACE product, the original PCR fragment, and the 3` RACE product generated a contiguous DNA sequence. To ensure that the product of the alignment represented one contiguous cDNA, EcoRI- linkered primers corresponding to the extreme 5` and 3` ends of the sequence were used to PCR the cDNA in one piece from human brain cDNA. The PCR product was subcloned into the EcoRI site of pUC19 and sequenced.
The ORF encoding the processed form of human
FKBP13 (hFKBP13) fused to 10 histidine residues was generated by PCR
using the sense oligonucleotide
AGATATACCATGGGCCATCATCATCATCATCATCATCATCATCACAGCAGCGGCCATATCGACGACGACGACAAGACGGGGGCCGAGGGCAAAAGG
and the antisense oligonucleotide AACCTTGGATCCTTACAGCTCAGTTCGTCGCTC.
The PCR product was digested with NcoI and BamHI and
cloned between the NcoI and BamHI sites of pET3d, and
the resulting plasmid was transformed into BL21(DE3) cells. Protein
expression and cell lysis was as described for hFKBP25. hFKBP13 was
applied to a nickel nitrilotriacetic acid (Qiagen) affinity column (1
cm 5 cm) and washed, and the pure protein was eluted with 250
mM imidazole (pH 7.0) according to the manufacturer's
instructions. The ORF encoding human FKBP52 (hFKBP52) was generated by
PCR using the sense oligonucleotide
AATTGTCGACCATATGACAGCCGAGGAGATGAAGGCG and the antisense oligonucleotide
AATTCTCGAGCTATGCTTCTGTCTCCACCTGAGA. The product was digested with NdeI and XhoI and cloned into pET15b (Novagen), a
polyhistidine fusion vector. hFKBP52 was expressed and purified as
described for hFKBP13 above. That recombinant hFKBP13 and hFKBP52 are
fully active was confirmed by their complete binding to FK506-Sepharose
and by their peptidyl-prolyl isomerase activities, which were in accord
with published values (for review, see (17) ). hCyPA was
expressed and purified to homogeneity as described
previously(18) . hFKBP13 and hFKBP52 were stored at -70
°C, and hCyPA was stored at 4 °C.
The high affinity
[H]ryanodine binding site density in cardiac and
skeletal muscle SR was determined by Scatchard analysis of
[
H]ryanodine binding isotherms as described
previously(6) . Because the native CRC contains one high
affinity binding site for ryanodine, the B
value
is proportional to the concentration of CRC present in SR. The
stoichiometry of FKBP per CRC was calculated directly from the ratio of B
values for
[
H]dihydro-FK506 and
[
H]ryanodine binding as described previously (6) .
Figure 2: Amino acid and nucleotide sequence of human FKBP12.6. The nucleotide sequence of the cDNA encoding human FKBP12.6 and the translated open reading frame are shown. The amino acid sequence is identical to that of bovine FKBP12.6(13) . Nucleotide numbering is with respect to the first base of the initiator methionine. Where two numbers are present, the lower number is the amino acid position, and the upper number is the nucleotide position. This sequence has been deposited in the Genome Sequence Data Base, the EMBL Data Library, the DNA Data Bank of Japan, and the NCBI under the accession number L37086.
Figure 3:
Steady-state hFKBP12.6 and hFKBP12 mRNA
levels in various human tissues and regions of the human brain.
Northern blots (Clontech) containing, per lane, 2 µg of
poly(A) RNA from various tissues or anatomically
distinct regions of the human brain were probed with the
P-labeled, randomly primed cDNAs encoding hFKBP12.6 (panels A, B, and E), hFKBP12 (panel
F), or
-actin (panels C, D, and G). Panels C and D are the actin controls
for panels A and B, respectively. Panel G is
the actin control for panels E and F, the same blot
probed with hFKBP12.6 and FKBP12, respectively. Arrows show
the locations of molecular weight markers in kilobase (kb)
pairs. Hybridizations were performed under high stringency conditions
and were washed according to the manufacturer's conditions. The
mRNA sources are as follows: lane 1, heart; lane 2,
brain; lane 3, placenta; lane 4, lung; lane
5, liver; lane 6, skeletal muscle; lane 7,
kidney; lane 8, pancreas; lane 9, spleen; lane
10, thymus; lane 11, prostate; lane 12, testis; lane 13, ovary; lane 14, intestine; lane 15,
colon; lane 16, peripheral blood lymphocyte; lane 17,
amygdala; lane 18, caudate nucleus; lane 19, corpus
collosum; lane 20, hippocampus; lane 21,
hypothalamus; lane 22, substantia nigra; lane 23,
subthalamic nucleus; and lane 24, thalamus. The exposure times
were as follows: panels A, B, and E, 4 days; panel F, 12 h; and panels C, D, and G, 2 h.
Figure 4: Purity of recombinant hFKBP12 and hFKBP12.6. Five µg of purified bacterially-expressed hFKBP12 (lane 2) and hFKBP12.6 (lane 3) were subjected to SDS-PAGE on a 16% gel (Novex). The following proteins (and their molecular weights) were used as standards (lane 1): phosphorylase b, 106,000; bovine serum albumin, 80,000; ovalbumin, 49,500; carbonic anhydrase, 32,500; soybean trypsin inhibitor, 27,500; and lysozyme, 18,500.
The catalytic
efficiency (k/K
) of hFKBP12
toward peptidyl-prolyl substrates correlates strongly with the
hydrophobicity of the amino acid immediately preceding the proline (37) . This contrasts with the promiscuous peptidyl-prolyl
isomerase substrate specificity observed with CyPA, the binding protein
for the structurally unrelated immunosuppressive drug, CsA. We compared
the abilities of purified recombinant hFKBP12.6 and hFKBP12 to catalyze
the isomerization to the trans form of tetrapeptides of the
general structure N-succinyl-Ala-Xaa-cis-Pro-Phe-p-nitroanilide
where Xaa is any one of 12 amino acids (Table 1). hFKBP12.6
exhibits substrate preferences similar, but not identical, to those
observed for hFKBP12. As with hFKBP12, substrates in which a
hydrophobic amino acid precedes proline are greatly preferred by
hFKBP12.6. For both FKBPs, the most reactive substrates have Leu, Ile,
Phe, or Nle at the Xaa position, while the least reactive substrate has
Gly at the Xaa position. With most of the tetrapeptide substrates
tested, the catalytic efficiency of hFKBP12.6 is roughly 2-fold lower
than that observed with hFKBP12. When Xaa is Val or Nle, the catalytic
efficiencies of hFKBP12 and hFKBP12.6 are about equal. Only with the
His-Pro substrate does hFKBP12.6 exhibit more reactivity than hFKBP12.
Figure 5: FKBP12.6 is associated with the RyR of canine heart SR. Samples of canine skeletal muscle terminal cisternae of SR (lanes 1-3) or cardiac SR (lanes 4-6) were analyzed by Western blot analysis. Samples were loaded in the absence(-) or presence (+) of either 30 ng of hFKBP12 (lanes 2 and 5) or 30 ng of hFKBP12.6 (lanes 3 and 6). Lanes 7 and 8 were loaded with 30 ng of hFKBP12 and 30 ng of FKBP12.6, respectively. The position of molecular weight standards, the bromphenol blue dye front (D) and the top of the resolving gel (T) are indicated to the left of the figure. The positions of bands corresponding to hFKBP12 and hFKBP12.6 are indicated at right. All other immunoreactive bands in lanes 1-6 are nonspecific because they are also observed in the absence of primary antibody. Cardiac SR was isolated in the presence of 0.6 M KCl, which is responsible for the band broadening (toward the bottom of the gel) and for the slightly slower mobility of both hFKBP12 and hFKBP12.6 in lanes 4-6.
Confirmation that FKBP12.6 is associated with the cardiac muscle RyR was obtained by amino-terminal sequencing of FKBP-C obtained from purified canine cardiac RyR preparations. FKBP-C was stripped from purified cardiac RyR with FK506 and separated from the RyR by hydroxyapatite chromatography. Amino-terminal sequencing of the purified protein gave the eleven amino acid sequence GVEIETISXGD, identical to the amino-terminal sequence of both bovine and human FKBP12.6 and different in two amino acids from the 11 amino-terminal amino acids of both bovine and human FKBP12, GVQVETISPGD. The observations that the RyR purified from canine heart is associated with a protein that co-migrates with FKBP12.6 on denaturing gels and has the same amino-terminal sequence as both bovine and human FKBP12.6 indicates that FKBP12.6 is specifically associated with the canine heart RyR. In the cytosol of dog heart and in canine skeletal muscle TC, only FKBP12 has been detected(32) , indicating that the interaction between FKBP12.6 and the heart RyR is specific and not due to the absence of FKBP12 in heart muscle.
The observation that FKBP12.6 associates specifically with the cardiac CRC makes it likely that FKBP12.6 modulates channel gating of the cardiac isoform in a manner similar to that observed for modulation of the skeletal muscle RyR-1 by FKBP12. Thus, the few amino acid differences between FKBP12 and FKBP12.6 have important consequences for channel binding specificity. We have expanded our characterization of hFKBP12.6 and have performed a pharmacological comparison of hFKBP12.6 and hFKBP12 both in vitro and in Jurkat cells in an effort to uncover differences between the two molecules that might help to explain their apparently different physiological roles.
The phosphatase assays were
designed to measure CaN inhibition by the various immunophilin-drug
complexes and to minimize any effect made by the equilibrium between
the complex and the free immunophilin and drug molecules. Therefore,
immunophilin-drug complex formation at a particular immunophilin or
drug concentration was maximized by having an excess of one component.
In one set of assays (Fig. 6A), drugs (FK506 or CsA)
were titrated in the presence of a constant high concentration (50
µM) of immunophilin to insure that most of the added drug
would be bound. In a second set of assays (Fig. 6B) the
immunophilins were titrated in the presence of a high concentration (50
µM) of drug to insure saturation of added binding protein.
As expected, both types of assays gave similar results. Irrespective of
which component is titrated, the IC values (legend to Fig. 6) obtained for CaN inhibition by a particular
immunophilin-drug complex are similar to one another. Both the drug and
immunophilin titrations (Fig. 6, A and B,
respectively) demonstrate that the FK506 complexes with hFKBP12.6 and
hFKBP12 are equipotent to one another and to the yFKBP12
FK506
complex as CaN inhibitors. As a control, and to further validate the
assay, the ability of the hCyPA
CsA complex to inhibit CaN was
measured. In agreement with observations that, by several criteria, CsA
is 10-100-fold less potent than FK506 (for review, see (40) ), the hCyPA
CsA complex was about 15-fold less
active than the hFKBP12
FK506 complex as a CaN inhibitor (Fig. 6, A and B). The remaining
hFKBP
FK506 complexes are very poor CaN inhibitors. hFKBP25 is
unable to inhibit CaN at even the highest drug and immunophilin
concentrations tested. The hFKBP13
FK506 and hFKBP52
FK506
complexes are very weak inhibitors of CaN activity. Phosphatase
inhibition by the latter two complexes is observed at immunophilin-drug
concentrations unlikely to be attained within most cells. Thus, the
hFKBP13
FK506 and hFKBP52
FK506 complexes may not make
significant contributions to CaN-dependent immunosuppression or
toxicity.
Figure 6:
hFKBP12.6 and hFKBP12 are equipotent
mediators of calcineurin inhibition by both FK506 and `818. The FK506
and `818 complexes with the known human FKBPs, with yFKBP12, and with
hCyPA were tested for their ability to inhibit CaN phosphatase
activity. Incubation and assay conditions are described under
``Materials and Methods.'' Results are plotted as the
percentage of the uninhibited control where no drug was present and in
which 3 nM CaN dephosphorylates 14.3 pmol of RII
phosphopeptide/min. Each data point represents an average of two
experiments. Panel A, increasing concentrations of FK506 (or
CsA, when CyPA was the immunophilin were added to CaN, CaM,
MgCl, CaCl
, and 50 µM immunophilin. The IC
values (in parentheses) of FK506
(or CsA when hCyPA was the immunophilin) complexed with the indicated
immunophilin are as follows:
, hFKBP12 (14.3 nM);
, hFKBP12.6 (10.6 nM);
, yFKBP12 (7.4
nM);
, hFKBP13 (partial inhibition);
, hFKBP25 (no
inhibition);
, hFKBP52 (partial inhibition);
, hCyPA (301
nM). Panel B, increasing concentrations of
immunophilin were added to CaN, CaM, MgCl
,
CaCl
, and 50 µM FK506 (or 50 µM CsA, when hCyPA was the immunophilin). The IC
values
(in parentheses) of the indicated immunophilin (symbols are as in panel A) complexed with FK506 (or CsA in the case of hCyPA)
are as follows: hFKBP12 (7.6 nM); hFKBP12.6 (8.6 nM);
yFKBP12 (10.4 nM); hFKBP13 (partial inhibition); hFKBP25 (no
inhibition); hFKBP52 (partial inhibition); hCyPA (125 nM). Panel C, as described in panel A except that `818 was
titrated. The IC
values (in parentheses) of `818 complexed
with the indicated FKBP (symbols are as in panel A) are as
follows: hFKBP12 (2.9 µM); hFKBP12.6 (3.1
µM); yFKBP12 (94 nM); hFKBP13 (no inhibition);
hFKBP25 (no inhibition); hFKBP52 (no inhibition). Panel D,
immunophilins were titrated as described in panel B except in
the presence of 50 µM `818. The IC
values (in
parentheses) of the indicated FKBP (symbols are as in panel A)
complexed with `818 are as follows: hFKBP12 (3.4 µM);
hFKBP12.6 (2.6 µM); yFKBP12 (176 nM); all other
FKBPs (no inhibition).
In noteworthy contrast to hFKBP12, the
yFKBP12 complex with `818 is a potent CaN inhibitor(5) .
Surrounding FK506 on the solvent-exposed surface of the complex are
approximately 26 amino acids that are likely to be close to CaN.
Throughout these 26 residues, there are 10 differences between yeast
and human FKBP12. Because the three-dimensional structures of the yeast
and human FKBP12`818 complexes are almost identical, among the 10
changes in yFKBP12 there must be amino acids that directly interact
with CaN, thereby compensating for `818(42) . These amino acids
in yFKBP12 in some way neutralize the effects of the hydroxyl group at
C-18(5) . In the absence of a crystal structure for the
FKBP12
FK506
CaN complex, `818 is a useful pharmacological
probe for helping to identify FKBP residues that might interact with
CaN.
Of the 18 amino acid differences between hFKBP12.6 and hFKBP12,
two residue changes in hFKBP12.6 (Arg and Val
in Fig. 2), are among those 26 surface residues that
surround the drug on the face of the complex. Because `818 uncovered
differences between yeast and human FKBP12 not observed with FK506, we
believed it might also uncover differences between hFKBP12 and
hFKBP12.6. Therefore, the FKBP12.6
`818 complex as well as the
`818 complexes with all other hFKBPs and yFKBP12 were tested for CaN
inhibition. As before, two versions of the assay, titrating drug (Fig. 6C) and titrating immunophilin (Fig. 6D), were performed with both assays giving
similar IC
values (legend to Fig. 6). As described
previously, yFKBP12 is a surprisingly potent CaN inhibitor when
complexed with `818(5) , albeit 24-fold less potent than when
complexed with FK506 (Fig. 6). When sufficiently high
concentrations (µM) of immunophilin are present, we find
that the hFKBP12
`818 complex can inhibit CaN, in contrast to
results from previous assays (4, 5) where the
submicromolar hFKBP12 concentrations used were insufficient to bind the
micromolar amounts of `818 required for detectable inhibitory activity.
The `818 complex with hFKBP12.6 is equipotent to the hFKBP12
`818
complex as a CaN inhibitor. However, both complexes are
200-400-fold poorer CaN inhibitors than the FK506 versions of the
complexes. Therefore, unlike yFKBP12, none of the amino acid
differences in hFKBP12.6 relative to hFKBP12 are able to compensate for
the hydroxyl group of `818. In the presence of `818, none of the other
FKBPs can inhibit CaN, corroborating the observations made with FK506.
We performed an assay similar to the one
described(12) , but to increase sensitivity, we incorporated an
important modification. Overexpression of the catalytic subunit of CaN
(CaN) is known to render activated Jurkat cells 4-5-fold
less sensitive to the effects of FK506 and
CsA(3, 43) . Our modification was to overexpress the
cDNAs encoding both the catalytic (CaN
) and regulatory (CaN
)
subunits of CaN by transient transfection in Jurkat cells using the
mammalian expression vector pcDL-SR
296 (SR
)(23) .
Protein overexpression was confirmed by Western blot analysis comparing
extracts from transfected and nontransfected cells (Fig. 7), and
drug sensitivity was quantitated by measuring
-galactosidase
production from a co-transfected reporter plasmid, pIL2.Gal, containing
the IL-2 promoter fused to the
-galactosidase reporter gene.
Overexpression of both CaN
and CaN
rendered the cells
insensitive to the effects of up to 10 nM FK506, almost 1000
times the normal IC
, 0.012 nM (Fig. 8A). There are two possible explanations for the
drug insensitivity generated by CaN overexpression. Either the CaN is
ectopically expressed in a subcellular compartment, separating it from
the hFKBP12
FK506 complex, or the CaN levels have exceeded the
FKBP12 levels such that, even at the highest drug concentrations, there
is sufficient free CaN available to participate in the signaling
pathway. If the second explanation is correct, then co-expression of
hFKBP12 will revert the cells to drug sensitivity and we will have
generated an assay with a greatly amplified readout relative to the
2-3-fold shift in IC
observed in the previous
assay(12) . To test between these alternatives, Jurkat cells
overexpressing CaN
and CaN
were co-transfected with
expression constructs encoding the FKBPs tested in the previous assay
(hFKBP12, hFKBP13, and hFKBP25) (12) . Overexpression of
hFKBP12, confirmed by Western blotting (Fig. 7), reestablishes
the FK506-sensitivity of the cells (Fig. 8A).
Reflecting the greatly increased amount of CaN that must be inhibited,
the IC
(0.26 nM) has shifted 20-fold relative to
nontransfected cells (0.012 nM). This result indicates that
CaN-overexpression is cytosolic and that hFKBP12 mediates
FK506-sensitivity in a T-cell line, thereby validating our assay. In
contrast, sensitivity to FK506 is not recovered upon hFKBP25
overexpression (Fig. 8A), confirming the results of
Bram et al.(12) and our result that the
hFKBP25
FK506 complex cannot inhibit CaN in vitro. We
find that hFKBP13 has some ability to mediate the inhibitory effects of
FK506, consistent with our observation (see Fig. 6) and with the
observations of others(44, 45) that the
hFKBP13
FK506 complex can inhibit, albeit weakly, CaN phosphatase
activity.
Figure 7:
Western blot analysis of immunophilin and
calcineurin expression in transfected Jurkat cells. Cytosolic extracts
from nontransfected (lane 1) and transfected (lane 2)
Jurkat cells are compared with standards consisting of 1 ng (lane
3), 3 ng (lane 4), 10 ng (lane 5), 30 ng (lane 6), 300 ng (lane 7), and 1 µg (lane
8) of the corresponding bacterially produced, purified proteins: A, hFKBP12; B, hFKBP12.6; C, hFKBP13; D, hFKBP25; E, hFKBP52; F, murine CaN; G, hCyPA; and H, yFKBP12. The amount of hFKBP13 in
the cytosolic extract (shown here) of the transfected cells is probably
an underestimate of the amount actually produced because it can
associate with membranes. For experimental details and for details of
the antibodies used, see ``Materials and
Methods.''
Figure 8:
hFKBP12.6 is equipotent to hFKBP12 at
mediating the FK506-sensitivity of the IL-2 promoter in transfected
Jurkat cells. Activities are plotted as the percent of
-galactosidase activity in lysates of activated cells that were
not treated with drug. The amount of
-galactosidase produced in
the absence of drug (No-Drug Controls) varied by less than 15%
among the various transfectants, thereby demonstrating that
transfection efficiencies were equivalent and uniform. Each data point
represents the mean of three experiments with a standard error of less
than 10%. Panel A, FK506-insensitivity caused by
overexpression of the catalytic (CaN
) and regulatory (CaN
)
subunits of CaN is reversed by overexpression of hFKBP12. Jurkat cells
transfected with pIL2.Gal were mock-transfected or co-transfected with
the SR
expression vector containing the indicated cDNAs and
activated in the presence of FK506, the amount of
-galactosidase
was measured, and the IC
values (in parentheses) of FK506
were determined: ⊞, mock transfection (0.012 nM);
, SR
vector only (0.012 nM);
, CaN
and
CaN
(no inhibition);
, hFKBP12, CaN
, and CaN
(0.26
nM);
, hFKBP13, CaN
, and CaN
(partial
inhibition);
, hFKBP25, CaN
, and CaN
(no inhibition). Panel B, FK506-insensitivity caused by CaN overexpression is
reversed by overexpression of hFKBP12.6. The expressed proteins and the
IC
values (in parentheses) for FK506 are as follows:
, SR
vector only (replotted from panel A; 0.012
nM);
, CaN
and CaN
(replotted from panel
A; no inhibition);
, hFKBP12.6, CaN
, and CaN
(0.18 nM);
, hFKBP52, CaN
, and CaN
(partial
inhibition);
, yFKBP12, CaN
and CaN
(0.37
nM). Panel C, effect of FKBP overexpression on the
IC
of `818. Jurkat cells transfected with pIL2.Gal were
mock-transfected or co-transfected with the SR
expression vector
containing the indicated cDNAs and activated in the presence of `818,
the amount of
-galactosidase was measured, and the IC
values (in parentheses) of `818 were determined. ⊞, mock
transfection (no inhibition);
, yFKBP12 (0.27 nM);
, hFKBP12 (2.7 nM);
, hFKBP12.6 (2.3
nM);
, hFKBP13 (no inhibition);
, hFKBP25 (no
inhibition);
, hFKBP52 (no inhibition). Panel D, CsA
insensitivity caused by CaN-overexpression is reversed by
overexpression of hCyPA. Jurkat cells transfected with pIL2.Gal were
mock-transfected or co-transfected with the SR
expression vector
containing the indicated cDNAs and activated in the presence of CsA,
the amount of
-galactosidase was measured, and the IC
(in parentheses) of CsA determined. ⊞, mock transfection
(0.57 nM);
, SR
vector only (0.45 nM);
, CaN
and CaN
(27.5 nM);
, hFKBP12,
CaN
, and CaN
(24.2 nM);
, hFKBP12.6,
CaN
, and CaN
(19.5 nM);
, hFKBP13, CaN
,
and CaN
(24.3 nM);
, hFKBP25, CaN
, and CaN
(24.1 nM);
, hFKBP52, CaN
, and CaN
(25.9
nM);
, hCyPA, CaN
, and CaN
(3.0
nM).
Finally, the ability of hFKBP12.6 to restore FK506
sensitivity in CaN-overexpressing Jurkat cells was examined. We also
tested hFKBP52 and yFKBP12 since the FK506 complexes with these FKBPs
have been characterized for CaN inhibition, in
vitro(5, 46) , but have not been tested in Jurkat
cells. Transfection of the CaN-overexpressing Jurkat cells with the
cDNA encoding hFKBP12.6 restored FK506-sensitivity (Fig. 8B). The IC of FK506 in the cells
overexpressing hFKBP12.6 is 0.18 nM, demonstrating that
hFKBP12.6 is equipotent to hFKBP12 at mediating the inhibitory effects
of FK506 upon CaN-dependent signaling events in Jurkat cells and
corroborating our result that the hFKBP12.6 and hFKBP12 complexes with
FK506 are equipotent CaN inhibitors in vitro. Also in
agreement with the CaN phosphatase assay, yFKBP12 is equipotent to both
hFKBP12 and hFKBP12.6 at mediating the inhibitory effects of FK506 in
Jurkat cells (Fig. 8B). In contrast, the FK506 complex
with hFKBP52 is only a weak inhibitor of signaling since, even at 10
µM drug concentrations, it is unable to completely block
IL-2 promoter activation (Fig. 8B).
Overexpression of
hFKBP12.6 renders activated Jurkat cells sensitive to `818 (Fig. 8C). Moreover, hFKBP12.6 and hFKBP12 are
equipotent in this assay, in agreement with their equal abilities to
mediate FK506-sensitivity in vivo and in accord with their
abilities, when complexed with `818, to inhibit CaN in vitro.
Further validation of these transfection experiments as an accurate
measure of the ability of FKBPs to mediate drug sensitivity was
obtained when the yFKBP12 expression plasmid was transfected into
Jurkat cells. IL-2 promoter activity in activated cells is strikingly
sensitive to the yFKBP12`818 complex (Fig. 8C),
again correlating with the results obtained in the CaN phosphatase
assay. Overexpression of hFKBPs 13, 25, or 52 had no effect on the `818
sensitivity of the IL-2 promoter, confirming that these FKBP complexes
with FK506-like drugs are ineffective inhibitors of the signaling
pathway to IL-2 gene transcription.
The CsA sensitivity of the IL-2
promoter in activated Jurkat cells was measured as a control.
Transfections with the cDNAs encoding the CaN and CaN
subunits rendered the promoter less sensitive but not insensitive to
CsA (Fig. 8D), which differs from the results obtained
with FK506. The inability to make the cells insensitive to CsA upon CaN
overexpression is due to naturally high CyPA levels in Jurkat cells (Fig. 7, row G). Scanning densitometry (data not shown)
of the bands in Fig. 7shows that the molar concentration of
endogenous CyPA in Jurkat cells is still 7-fold greater than CaN
,
even when the latter protein is overexpressed (Fig. 7, compare lanes F and G). Thus, in CaN-overexpressing cells,
the molarity of CaN does not exceed the natural molarity of hCyPA,
explaining why the cells cannot be rendered completely insensitive to
CsA. Nevertheless, the CaN-overexpressing cells are less sensitive to
CsA because there is more CaN to inhibit (Fig. 8D). The
decreased sensitivity to CsA is reversed 9-fold in the
CaN-overexpressing cells by co-transfection with the cDNA encoding
hCyPA (Fig. 8D) thereby confirming previous results (12) that hCyPA can mediate the inhibitory effects of CsA. The
FKBPs are specific for mediating the inhibitory effects of FK506 and
not of CsA because FKBP overexpression does not alter the
CsA-sensitivity of the IL-2 promoter (Fig. 8D).
Figure 9:
hFKBP12.6RAP binds to mTOR. Rat
brain extracts were incubated with GST, GST-hFKBP12.6, or GST-hFKBP12
coupled to GSH
agarose beads. Precipitations were performed
without drug(-) or in the presence of 10 µM RAP or
10 µM FK506 (FK). Precipitated proteins were
eluted, resolved by SDS-PAGE through a 8.75% gel, and subjected to
Western blotting. The blot was probed first with the anti-mTOR
antibody. The blot was stripped and reprobed with anti-CaN antibodies
that recognize all three CaN
isoforms. Panel A shows that
portion of the blot with bands immunoreactive with the anti-mTOR
antibody. Panel B shows that portion of the blot having bands
immunoreactive with the anti-CaN
antibodies. The molecular masses
(kDa) of the calibration standards are indicated at the left and lane numbers are indicated at the bottom. The arrow labeled mTOR indicates the protein that binds
to the GST-hFKBP12.6
RAP and GST-hFKBP12
RAP complexes. The arrow labeled CaN
shows the location of the 57-
and 61-kDa isoforms of the catalytic CaN
subunit.
We have cloned the cDNA encoding human FKBP12.6 and have characterized the expressed protein pharmacologically and physiologically. Physiologically, FKBP12.6 has a role distinct from that of FKBP12. FKBP12 is associated with RyR-1 of skeletal muscle SR, whereas FKBP12.6 is specifically associated with RyR-2 of cardiac muscle SR. Pharmacologically, FKBP12.6 is almost indistinguishable from FKBP12. FKBP12.6 is the only other FKBP family member equipotent to FKBP12 at inhibiting CaN in vitro and at mediating the FK506-sensitivity of a CaN-dependent signal transduction pathway. Moreover, when complexed with RAP, FKBP12.6, like FKBP12, binds mTOR.
The cardiac CRC (RyR-2) is a 565-kDa protomer 64% identical to RyR-1 (50, 51) . The hydropathy profiles and predicted
secondary structures of the cardiac and skeletal isoforms are virtually
identical(51) . Both are activated by Ca,
ATP, and caffeine; both are inactivated by Mg
and
ruthenium red; and both contain one high affinity and several low
affinity ryanodine binding sites(52) . Although morphologically
and functionally similar, the channels are not identical(52) .
We have shown that FKBP-C(32) , isolated from the canine
cardiac RyR, co-migrates with hFKBP12.6 on SDS-PAGE gels and has the
same 11-amino acid amino-terminal sequence as both bovine and human
FKBP12.6. Our finding that there are four FK506 binding sites per high
affinity ryanodine binding site in cardiac SR suggests that the
structure of the cardiac CRC can be represented as
(RyR-2)
(FKBP12.6)
, analogous to the structure
of the skeletal muscle CRC, (RyR-1)
(FKBP12)
.
Thus, the native CRC isoforms in heart and skeletal muscle SR are
further distinguished from one another in that different FKBP isoforms
comprise a portion of their structures. The structural and functional
similarities between FKBP12.6 and FKBP12 and between the cardiac and
skeletal muscle RyR isoforms, suggests that the role of FKBP12.6 in the
native cardiac CRC is similar to the role of FKBP12 in the skeletal
muscle CRC.
The stoichiometry of four molecules of FKBP12.6 per
native tetrameric CRC obtained by
[H]dihydro-FK506 binding isotherms relies on the
assumption that the ryanodine receptor is the predominant or only SR
protein that binds FKBP12.6. Recent studies confirm that this is the
case. Endogenous FKBP of cardiac SR was exchanged with the GST-FKBP12.6
fusion protein using exchange methodology developed for the skeletal
muscle RyR(9) . The TC was then solublized with CHAPS, and
protein complexes with the GST-FKBP12.6 fusion protein were affinity
purified on a GST-Sepharose affinity column. RyR-2 was the predominant
protein in the SR that was tightly bound to GST-FKBP12.6. (
)
In the presence of drug, the abilities of several human
FKBPs to inhibit CaN in vitro and a CaN-dependent signaling
pathway in cells have been compared. The abilities of the
immunophilin-drug complexes to inhibit CaN and their abilities to block
IL-2 transcription correlate precisely. The FK506 complexes with FKBP13
and FKBP52, weak CaN inhibitors in vitro, are weak inhibitors
of IL-2 promoter activity when the proteins are overexpressed in Jurkat
cells. This correlation extends to the yFKBP12`818 complex, a
more potent CaN inhibitor and a more potent inhibitor of IL-2 promoter
stimulation than the `818 complexes with hFKBP12 or hFKBP12.6. Thus,
our results support the proposed mechanism of action of FK506 in which
CaN inhibition blocks IL-2 transcription, thereby preventing T-cell
activation(54) . The RAP complexes with hFKBP12.6 and hFKBP12
exhibit similar but not identical properties. Qualitatively, the
hFKBP12
RAP complex binds more mTOR than the hFKBP12.6
RAP
complex, suggesting that hFKBP12 plays a greater role in mediating
RAP's antiproliferative effects in human cells.
The adverse
side effects of FK506 immunotherapy include nephrotoxicity,
diabetogenicity, the development of lymphoproliferative disorders,
expressive aphasia, seizures, coma, drowsiness, lethargy, tremors, and
aggressiveness(36, 55) . Toxicities associated with
RAP treatment in non-rodent mammals include vomiting, diarrhea,
thrombocytopenia, and gastrointestinal ulceration(56) . Because
inhibition of IL-2 promoter activity in T-cells is a convenient measure
of the in vivo potentials of FKBPFK506 complexes to
inhibit a CaN-dependent signaling pathway, our data reflect the impact
that the various FKBP
FK506 complexes can have upon a
physiological process involving CaN. The response of any single cell to
FK506 or RAP will depend upon several factors including 1) the
expression levels of the immunophilins mediating drug action; 2) the
abilities of the immunophilin-drug complexes to interact with their
immediate targets; 3) the concentration of FK506 or RAP that gets into
the cell; 4) the concentration of CaN or mTOR in the cell; and 5) the
function of the downstream substrates of CaN or mTOR and their
importance to the cell. Thus, the therapeutic and toxic side effects of
FK506 and RAP are likely due to the formation of multiple
FKBP
drug complexes with varying affinities for their
pharmacologic target proteins, CaN and mTOR. Although nothing is known
about mTOR substrates, CaN substrates that are candidate proteins
responsible for the deleterious effects of FK506 at recognized sites of
toxicity include the Na
,K
-ATPase in
the brain and kidney (57) and nitric oxide synthase in the
brain (58) . There have been no reports of FK506 toxicity that
would implicate the CRC. Because only a portion of the cellular FKBP
pool is bound by FK506 at immunosuppressive doses, the cytosolic FKBP
in cardiac and skeletal muscle may act as a buffer shielding the CRC
from the effects of the drug.
The design of one of our assays (Fig. 8), reversing the effects of CaN-overexpression,
underscores the importance of the cellular FKBP:CaN ratio in
establishing FK506-sensitivity. FKBP12 is both ubiquitous and abundant (59) and a survey of 15 different rat tissues documented
FKBP/CaN ratios ranging from 13 to 343(60) . The lowest
FKBP/CaN ratios were found in seven anatomically distinct regions of
the brain, a reflection of the great abundance (61) and
probable importance, of CaN there. FKBP/CaN ratios in the thymus and
spleen are among the highest, approximately 200 and 100,
respectively(60) . If the FKBP/CaN ratios in spleen and thymus
reflect ratios to be found in lymphocytes, then the Law of Mass Action
dictates that, in the presence of FK506, a greater proportion of CaN
will be inhibited in lymphocytes than in brain cells (even assuming
that the drug is distributed equally to the brain, which it is not). In
both Jurkat and murine T-cells, the intracellular concentration of FKBP
is 6-7 µM with only 3-5% of the FKBP pool
bound by FK506 at the IC for inhibition of cellular
activation(53) . To decrease neurotoxic effects associated with
FK506 therapy, one strategy would be to design FK506 analogs with
decreased affinity for FKBP, thereby allowing equilibrium to relieve
toxicity. With decreased affinity for FKBP, less FKBP
drug complex
would be formed to inhibit the high levels of CaN in brain. In lymphoid
cells, the high FKBP/CaN ratio would compensate for the lower affinity
of the novel analog for FKBP, thereby maintaining the immunosuppressive
efficacy of the drug. Similar considerations apply to the toxic effects
associated with RAP therapy.
The substrate preferences exhibited by FKBP12.6 and FKBP12 in the peptidyl-prolyl isomerase assay overlap almost completely. Therefore, our observation that FKBP12.6 and FKBP12 are physiologically associated with distinct RyR isoforms may be unrelated to peptidyl-prolyl isomerase activity. Both in the presence and absence of drug, the biochemical and cellular read-outs used in our study have demonstrated that FKBP12.6 is highly similar to FKBP12 and that, where it is abundant, FKBP12.6 will be an important mediator of the inhibitory effects of FK506 and RAP. Our inability to uncover any significant biochemical or pharmacological differences between the two immunophilins that might account for their overlapping, and yet distinct, physiological roles suggests further complexities among the FKBPs remaining to be understood.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L37086[GenBank].