Locations of calmodulin and FK506-binding protein on the three-dimensional architecture of the skeletal muscle ryanodine receptor

Isolated skeletal muscle ryanodine receptors (RyRs) complexed with the modulatory ligands, calmodulin (CaM) or 12-kDa FK506-binding protein (FKBP12), have been characterized by electron cryomicroscopy and three-dimensional reconstruction. RyRs are composed of 4 large subunits (molecular mass 565 kDa) that assemble to form a 4-fold symmetric complex that, architecturally, comprises two major substructures, a large ( approximately 80% of the total mass) cytoplasmic assembly and a smaller transmembrane assembly. Both CaM and FKBP12 bind to the cytoplasmic assembly at sites that are 10 and 12 nm, respectively, from the putative entrance to the transmembrane ion channel. FKBP12 binds along the edge of the square-shaped cytoplasmic assembly near the face that interacts in vivo with the sarcolemma/transverse tubule membrane system, whereas CaM binds within a cleft that faces the junctional face of the sarcoplasmic reticulum membrane at the triad junction. Both ligands interact with a domain that connects directly to a cytoplasmic extension of the transmembrane assembly of the receptor, and thus might cause structural changes in the domain which in turn modulate channel gating.     

Cardiac defects and altered ryanodine receptor function in mice lacking FKBP12

FKBP12, a cis-trans prolyl isomerase that binds the immunosuppressants FK506 and rapamycin, is ubiquitously expressed and interacts with proteins in several intracellular signal transduction systems. Although FKBP12 interacts with the cytoplasmic domains of type I receptors of the transforming growth factor-beta (TGF-beta) superfamily in vitro, the function of FKBP12 in TGF-beta superfamily signalling is controversial. FKBP12 also physically interacts stoichiometrically with multiple intracellular calcium release channels including the tetrameric skeletal muscle ryanodine receptor (RyR1). In contrast, the cardiac ryanodine receptor, RyR2, appears to bind selectively the FKBP12 homologue, FKBP12.6. To define the functions of FKBP12 in vivo, we generated mutant mice deficient in FKBP12 using embryonic stem (ES) cell technology. FKBP12-deficient mice have normal skeletal muscle but have severe dilated cardiomyopathy and ventricular septal defects that mimic a human congenital heart disorder, noncompaction of left ventricular myocardium. About 9% of the mutants exhibit exencephaly secondary to a defect in neural tube closure. Physiological studies demonstrate that FKBP12 is dispensable for TGF-beta-mediated signalling, but modulates the calcium release activity of both skeletal and cardiac ryanodine receptors.     

Crystal structure of cytoplasmic Escherichia coli peptidyl-prolyl isomerase: evidence for decreased mobility of loops upon complexation

Ryanodine receptors (RyRs), a class of intracellular calcium release channels, are the largest ion channels known. Recently, cryoelectron microscopy and image reconstructions of isolated receptors have shown that most of the protein mass forms a porous, multidomain cytoplasmic assembly. Evidence is mounting that suggests that the cytoplasmic assembly communicates with the transmembrane regions over distances of 100 or greater. RyRs are centrally important in excitation-contraction coupling, which occurs at specialized regions where the sarcoplasmic reticulum, containing the RyRs, and the plasma membrane/transverse-tubule system form junctions. Numerous proteins are present at these junctions, some of which interact directly with the RyR.     

Specific binding of tetratricopeptide repeat proteins to the C-terminal 12-kDa domain of hsp90

The molecular chaperone hsp90 in the eukaryotic cytosol interacts with a variety of protein cofactors. Several of these cofactors have protein domains containing tetratricopeptide repeat (TPR) motifs, which mediate binding to hsp90. Using a yeast two-hybrid screen, the 12-kDa C-terminal domain of human hsp90alpha (C90) was found to mediate the interaction of hsp90 with TPR-containing sequences from the hsp90 cofactors FKBP51/54 and FKBP52. In addition, the mitochondrial outer membrane protein hTOM34p was identified as a TPR-containing putative partner protein of hsp90. In experiments with purified proteins, the TPR-containing cofactor p60 (Hop) was shown to form stable complexes with hsp90. A deletion mutant of hsp90 lacking the C90 domain was unable to bind p60, whereas deletion of the approximately 25-kDa N-terminal domain of hsp90 did not affect complex formation. Both p60 and FKBP52 bound specifically to the C90 domain fused to glutathione S-transferase and competed with each other for binding. In reticulocyte lysate, the C90 fusion protein recognized the TPR proteins p60, FKBP52, and Cyp40. Thus, our results identify the C90 domain as the specific binding site for a set of hsp90 cofactors having TPR domains.     

FK-binding protein is associated with the ryanodine receptor of skeletal muscle in vertebrate animals

The ryanodine receptor/calcium release channel (RyR1) of sarcoplasmic reticulum from rabbit skeletal muscle terminal cisternae (TC) contains four tightly associated FK506-binding proteins (FKBP12). Dissociation and reconstitution studies have shown that RyR1 can be modulated by FKBP12, which helps to maintain the channel in the quiescent state. In this study, we found that the association of FKBP with RyR1 of skeletal muscle is common to each of the five classes of vertebrates. TC from skeletal muscle representing animals from different vertebrates, i.e. mammals (rabbit), birds (chicken), reptiles (turtle), fish (salmon and rainbow trout), and amphibians (frog), were isolated. For each, we find the following: 1) FKBP12 is localized to the TC (there are four FKBP binding sites/ryanodine receptor); 2) soluble FKBP exchanges with the bound form on RyR1 of TC; 3) release of FKBP from terminal cisternae by drug (FK590) treatment leads to a significant reduction in the net calcium loading rate, consistent with channel activation (the calcium loading rate is restored to the control value by reconstitution with FKBP12); and 4) RyR1 of skeletal muscle TC can bind to and exchange with either FKBP12 or FKBP12.6 (FKBP12.6 is the novel FKBP isoform found selectively associated with RyR2 of dog cardiac sarcoplasmic reticulum). We conclude that FKBP is an integral part of the RyR1 of skeletal muscle in each of the classes of vertebrate animals. The studies are consistent with a role for FKBP in skeletal muscle excitation-contraction coupling.     

Coupled gating between individual skeletal muscle Ca2+ release channels (ryanodine receptors)

Excitation-contraction coupling in skeletal muscle requires the release of intracellular calcium ions (Ca2+) through ryanodine receptor (RyR1) channels in the sarcoplasmic reticulum. Half of the RyR1 channels are activated by voltage-dependent Ca2+ channels in the plasma membrane. In planar lipid bilayers, RyR1 channels exhibited simultaneous openings and closings, termed "coupled gating." Addition of the channel accessory protein FKBP12 induced coupled gating, and removal of FKBP12 uncoupled channels. Coupled gating provides a mechanism by which RyR1 channels that are not associated with voltage-dependent Ca2+ channels can be regulated.     

Age-related changes of sodium-dependent D-[3H]aspartate and [3H]FK506 binding in rat brain

We investigated age-related changes in excitatory amino acid transport sites and FK506 binding protein (FKBP) in 3-week-, and 6-, 12-, 18- and 24-month-old Fischer 344 rat brains using receptor autoradiography. Sodium-dependent D-[3H]aspartate and [3H]FK506 were used to label excitatory amino acid transport sites and immunophilin (FKBP), respectively. In immature rats (3-week-old), sodium-dependent D-[3H]aspartate binding was lower in the frontal cortex, parietal cortex, striatum, nucleus accumbens, whole hippocampus, thalamus and cerebellum as compared to adult animals (6-month-old), whereas [3H]FK506 binding was significantly lower in only the hippocampus, thalamus and cerebellum. 3[H]FK506 binding exhibited no significant change in the brain regions examined during aging. However, sodium-dependent D-[3H]aspartate binding showed a conspicuous reduction in the substantia nigra in 18-month-old rats. Thereafter, a significant reduction in sodium-dependent D-[3H]aspartate binding was found in the thalamus, substantia nigra and cerebellum in 24-month-old rats. Other regions also showed about 10-25% reduction in sodium-dependent D-[3H]aspartate binding. The results indicate that excitatory amino acid transport sites are more susceptible to aging process than immunophilin. Further, our findings demonstrate the conspicuous differences in the developmental pattern between excitatory amino acid transport sites and immunophilin in immature rat brain.     

Cryoelectron microscopy and image analysis of the cardiac ryanodine receptor

The three-dimensional structure of the cardiac muscle ryanodine receptor (RyR2) is described and compared with its skeletal muscle isoform (RyR1). Previously, structural studies of RyR2 have not been as informative as those for RyR1 because optimal conditions for electron microscopy, which require low levels of phospholipid, are destabilizing for RyR2. A simple procedure was devised for diluting RyR2 (in phospholipid-containing buffer) into a lipid-free buffer directly on the electron microscope grid, followed by freezing within a few seconds. Cryoelectron microscopy of RyR2 so prepared yielded images of sufficient quality for analysis by single particle image processing. Averaged projection images for RyR2, as well as for RyR1, prepared under the same conditions, were found to be nearly identical in overall dimensions and appearance at the resolution attained, approximately 30 A. An initial three-dimensional reconstruction of RyR2 was determined (resolution approximately 41 A) and compared with previously reported reconstructions of RyR1. Although they looked similar, which is consistent with the similarity found for the projection images, and with expectations based on the 66% amino acid sequence identity of the two isoforms, structural differences near the corners of the cytoplasmic assembly were observed in both two- and three-dimensional studies.     

The stimulation of neoangiogenesis in the ischemic human heart by the growth factor FGF: first clinical results

AtFKBP12 is an Arabidopsis cDNA that encodes a protein similar to the mammalian immunophilin, FKBP12. AtFKBP12 was used as 'bait' in a yeast 2-hybrid system to screen for cDNAs in Arabidopsis encoding proteins that bind to FKBP12. Two partial cDNAs were recovered encoding the C-terminus of a protein we have called Arabidopsis thaliana FKBP12 interacting protein 37 (AtFIP37). AtFIP37 is similar to a mammalian protein, FAP48, that also binds to FKBP12. The interaction between AtFKBP12 and AtFIP37 in the 2-hybrid system, as assessed by histidine auxotrophy and beta-galactosidase activity, was disrupted by FK506, but not by cyclosporin A, a drug that binds to cyclophilin A. AtFIP37 was also shown to bind in vitro to AtFKBP12 in GST-fusion protein binding assays. The binding was abolished by prior incubation of AtFKBP12 with FK506. These findings indicate that an Arabidopsis FKBP12 ortholog encodes a protein that binds FK506 and that the interaction between AtFKBP12 and AtFIP37 may involve the FK506 binding site of AtFKBP12. The interaction provides interesting new opportunities for controlling protein:protein interactions in vivo in plants.     

The cyclophilin component of the unactivated estrogen receptor contains a tetratricopeptide repeat domain and shares identity with p59 (FKBP59)

Using a rapid single-step affinity chromatography procedure we have isolated the unactivated estrogen receptor from bovine uterus. Results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analyses for protein extracts recovered from affinity chromatography of receptor cytosols, either preincubated or untreated with estradiol, suggest a component structure for the intact oligomeric receptor which includes hsp90, hsp70, p59, a 40-kDa cyclophilin-related protein, and an uncharacterized 22-kDa protein species. We have chemically determined the amino acid sequences of eight peptides derived from the 40-kDa component and now report the cloning and primary sequence of a cDNA encoding this protein, which is designated estrogen receptor-binding cyclophilin (ERBC). Homology analyses confirm that ERBC is a new member of the cyclophilin family and contains a C-terminal domain with significant sequence homology to an internal region of p59, a binding protein for the immunosuppressant FK506 (FKBP59). This conserved region includes a 3-unit tetratricopeptide repeat domain bounded at the C terminus by a putative calmodulin binding site. We propose that the tetratricopeptide repeat domain mediates the protein interaction properties of ERBC and p59. Both immunophilins may have important roles in receptor assembly and may represent a new category of ligand- and calcium-dependent modulators of protein function.     

Calcineurin-immunosuppressor complexes

Crystal structures of the Ser/Thr phosphatase calcineurin (protein phosphatase 2B) have recently been solved by X-ray crystallography, both in the free-protein state, and complexed with the immunophilin/immunosuppressant FKBP12/FK506. Core elements of the calcineurin phosphatase have been found to be similar to the corresponding elements of Ser/Thr phosphatase 1 and purple acid phosphatase. The structures provide a basis for understanding calcineurin inhibition by a ternary complex of immunophilin and immunosuppressant proteins.     

FK506 binding protein mutational analysis. Defining the surface residue contributions to stability of the calcineurin co-complex

The 12- and 13-kDa FK506 binding proteins (FKBP12 and FKBP13) are cis-trans peptidyl-prolyl isomerases that bind the macrolides FK506 (Tacrolimus) and rapamycin (Sirolimus). The FKBP12.FK506 complex is immunosuppressive, acting as an inhibitor of the protein phosphatase calcineurin. We have examined the role of the key surface residues of FKBP12 and FKBP13 in calcineurin interactions by generating substitutions at these residues by site-directed mutagenesis. All mutants are active catalysts of the prolyl isomerase reaction, and bind FK506 or rapamycin with high affinity. Mutations at FKBP12 residues Asp-37, Arg-42, His-87, and Ile-90 decrease calcineurin affinity of the mutant FKBP12.FK506 complex by as much as 2600-fold in the case of I90K. Replacement of three FKBP13 surface residues (Gln-50, Ala-95, and Lys-98) with the corresponding homologous FKBP12 residues (Arg-42, His-87, and Ile-90) generates an FKBP13 variant that is equivalent to FKBP12 in its affinity for FK506, rapamycin, and calcineurin. These results confirm the role of two loop regions of FKBP12 (residues 40-44 and 84-91) as part of the effector face that interacts with calcineurin.     

Protease resistance of syntaxin.SNAP-25.VAMP complexes. Implications for assembly and structure

A stable ternary complex formed with vesicle-associated membrane protein 2 (VAMP2) and plasma membrane proteins syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP-25) is proposed to function in synaptic vesicle exocytosis. To analyze the structural characteristics of this synaptic protein complex, recombinant binary (syntaxin 1A.SNAP-25), recombinant ternary, and native ternary complexes were subjected to limited trypsin proteolysis. The protected fragments, defined by amino-terminal sequencing and mass spectrometry, included a carboxyl-terminal region of syntaxin 1A, the cytoplasmic domain of VAMP2, and amino- and carboxyl-terminal regions of SNAP-25. Furthermore, separate amino- and carboxyl-terminal fragments of SNAP-25, when combined with VAMP2 and syntaxin 1A, were sufficient for stable complex assembly. Analysis of ternary complexes formed with full-length proteins revealed that the carboxyl-terminal transmembrane anchors of both syntaxin 1A and VAMP2 were protected from trypsin digestion. Moreover, the stability of ternary complexes was increased by inclusion of these transmembrane domains. These results suggest that the transmembrane domains of VAMP2 and syntaxin 1A contribute to complex assembly and stability and that amino- and carboxyl-terminal regions of SNAP-25 may function as independent domains.     

A 37-amino acid sequence in the skeletal muscle ryanodine receptor interacts with the cytoplasmic loop between domains II and III in the skeletal muscle dihydropyridine receptor

Interactions between the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (ryanodine receptor or RyR1) and the loop linking domains II and III (II-III loop) of the skeletal muscle L-type Ca2+ channel (dihydropyridine receptor or DHPR) are critical for excitation-contraction coupling in skeletal muscle. The DHPR II-III loop was fused to glutathione S-transferase- or His-peptide and used as a protein affinity column for 35S-labeled in vitro translated fragments from the N-terminal three-fourths of RyR1. RyR1 residues Leu922-Asp1112 bound specifically to the DHPR II-III loop column, but the corresponding fragment from the cardiac ryanodine receptor (RyR2) did not. The use of chimeras between RyR1 and RyR2 localized the interaction to 37 amino acids, Arg1076-Asp1112, in RyR1. The RyR1 922-1112 fragment did not bind to the cardiac DHPR II-III loop but did bind to the skeletal muscle Na+ channel II-III loop. The skeletal DHPR II-III loop double mutant K677E/K682E lost most of its capacity to interact with RyR1, suggesting that two positively charged residues are important in the interaction between RyR and DHPR.     

Molecular chaperones and subcellular trafficking of steroid receptors

Unliganded steroid receptors exist as heteromeric complexes comprised of heat shock and immunophilin proteins that associate either directly or indirectly with receptor carboxyl-terminal ligand-binding domains. Molecular chaperons, and other proteins associated with steroid receptors, play an important role in the maturation of receptors to a hormone-binding competent state. Steroid receptor-associated 90 and 70 kDa heat shock proteins, hsp90 and hsp70, respectively, have well established roles in protein folding in addition to participating in numerous subcellular trafficking pathways. In this review, we discuss the possible roles that molecular chaperons, such as hsp90, hsp70 and DnaJ proteins, have in steroid receptor trafficking within two distinct subcellular compartments, i.e. the cytoplasm and nucleus.     

Growth hormone-promoted tyrosyl phosphorylation of a 121-kDa growth hormone receptor-associated protein

Previous work in multiple cell types has shown that endogenous GH receptors, as well as the cloned liver GH receptor, associate with a tyrosine kinase. However, in SDS-PAGE gels of highly purified, kinase-active GH receptor preparations from 35S-labeled 3T3-F442A cells, only one broad band was detected corresponding to the molecular weight of the GH receptor rather than two bands which might be expected to result from a kinase-receptor heterocomplex. In the present study, a transfected Chinese hamster ovary (CHO) cell line (CHO4) that expresses an 84-kDa GH receptor rather than a 121-kDa GH receptor was used to examine whether the GH receptor might form a complex with a protein (e.g. tyrosine kinase) that comigrates on SDS-polyacrylamide gel electrophoresis gels with the endogenous GH receptor (M(r) 121,000) in 3T3-F442A cells. GH-GH receptor complexes were immunoprecipitated with anti-GH antibody from GH-treated CHO4 cells and incubated with [gamma-32P]ATP. 32P was incorporated into a 121-kDa protein as well as the 84-kDa GH receptor. Phosphorylation of both the 84-kDa GH receptor and the 121-kDa protein was on tyrosyl residues as determined by Western blotting with anti-phosphotyrosine antibody. The 121-kDa protein does not appear to bind GH. It was also not detected in the immunoprecipitate when cells had not been incubated with GH or when untransfected CHO cells were used. These findings suggest that in CHO4 cells, the 121-kDa protein is precipitated by the GH antibody because of its ability to form a complex with the GH receptor (p84). Western blot analysis of whole cell lysates using anti-phosphotyrosine antibody revealed that GH promotes the tyrosyl phosphorylation of a 121-kDa protein and several other proteins (p97, p42, p39) in a dose- and time-dependent fashion. Taken together, these findings are consistent with either p121 being the tyrosine kinase that complexes with the GH receptor and is activated in response to GH binding or with p121 forming a ternary complex with both the GH receptor and a tyrosine kinase and serving as a substrate of the GH receptor-associated tyrosine kinase.     

Molecular characterization of Treponema pallidum mcp2, a putative chemotaxis protein gene

The nucleotide sequence of the Treponema pallidum mcp2 gene was determined. mcp2 encodes a 45.8-kDa protein whose deduced amino acid sequence has significant homology with the C-terminal region of bacterial methyl-accepting chemotaxis proteins (MCPs). The Mcp2 N terminus lacks the hydrophobic transmembrane regions present in most MCPs. An Mcp2 fusion protein was strongly reactive with antibody (HC23) to the highly conserved domain of MCPs and with rabbit syphilitic serum. Antibody HC23 reacted with six T. pallidum proteins, including a 45-kDa protein that may correspond to Mcp2. This protein was present in the aqueous phase from T. pallidum cells that were solubilized with Triton X-114 and phase partitioned.