RICERCA ITALIANA     

Research program

Supramolecular complexes of sorcin in the generation and regulation of Calcium-dependent cellular functions

University Co-ordinator

Università degli Studi di SIENA - NEUROSCIENZE - ()

Research Unit Leader

Vincenzo Sorrentino

Description

The approximately 4000 amino acid large N-terminal domain of the RyR is located in the cytosolic compartment and is the site of occurrence of a number of protein–protein interactions and binding to channel modulators (Lehnart, 2007). RyR2 has been shown to be phosphorylated by PKA (ser 2030 and ser 2808 in RyR2) and to bind the protein phosphatases PP1 and PP2A through leucine–isoleucine zipper (LIZ) motifs located in regions 554-588, 1603-1631 and 3003-3039 in RyR2. RyR2 can also be phophorylated by Ca2+–calmodulin-dependent protein kinase II (CaMKII) (Ser 2814 in RyR2) (Wehrens et al., 2004; Yang et al., 2007; Ferrero et al., 2007). Additional RyR-binding proteins/regulators include calstabin/FKBP12, which stabilizes the channel closed state (binding residues 1–1937 and 3788–4967 in RyR2) (Masumiya et al., 2003; Zissimopoulos and Lai, 2005a, 2005b), calmodulin (binding residues 3581–3610), Homer (773–1783 in RyR1) and sorcin (for a review see Bers, 2004). Genetic studies have identified 69 mutations in RyR2 causing catecholaminergic polymorphic ventricular tachycardia (CPVT), a malignant stress-induced arrhythmia. Interestingly, these mutations can be clustered in four regions of the RyR2 gene (CPVT-I, 77–466; CPVT-II, 2246–2534; CPVT-III, 3778–4201; CPVT-IV, 4497–4959), which largely overlaps with clustering of RyR1 mutations linked with malignant hyperthermia and central core disease, strongly suggesting that these represent critical regions for the correct functioning of the channel (George et al., 2007)

According to what previously discussed, the proposed project will be mainly focused on the characterization of the interaction occurring between sorcin, RyRs and CK2. In particular, unit Sorrentino will be interested in the identification of the regions in RyR2 important or responsible for its interaction with sorcin, by using in vitro interaction experiments and surface plasmon resonance. In addition, the interaction between RyR3 and sorcin will be also investigated on the basis of evidence that sorcin binds to RyR3 (Colotti, Rossi, Chiancone and Sorrentino unpublished) and of the high sequence homology existing between RyR2 and RyR3 in their regulatory regions. This part of the project will be performed in collaboration with unit Colotti, who will proceed with the biochemical characterization of the interaction between sorcin and defined regions of RyR2/RyR3. In parallel, this unit will collaborate will unit Ruzzene who recently demontrated an interaction between sorcin and CK2, to verify whether the interaction with and a possibile phosphorylation by CK2 can affect the ability of sorcin to regulate RyR2. Furthermore, again in collaboration with Ruzzene, unit we will test the possibilità that RyR2 can be phosphorylated by CK2.


Topology of RyR-sorcin interaction:
Functional interaction between sorcin and RyRs has been extensively studied in the cardiac muscle. However, no data are currently available on the identification of the specific interaction site for sorcin in RyR2. In order to meet this question regions of the N-terminal/cytoplasmic domain of RyR2 will be cloned in vectors suitable for protein expression and in vitro interaction studies. The N-terminal region of RyR2 spans amino acids 1 to 4511 in the rabbit RyR2 sequence. We are planning to start with 8 clones covering about 550 aminoacids each according to available cloning sites. The selected regions of RyR2 will be tested for sorcin interaction using in vitro interaction studies.

a) GST-pull down experiments: The RyR2 regions of interest will be inserted into pcDNA3 vectors to perform in vitro transcription and translation (TNT) experiments. For convenience each region of the cytoplasmic portion of RyR2 will be cloned in frame with a myc-tag epitope to be detected in the pull-down experiments by commercially available monoclonal or polyclonal antibodies. Alternatively the peptides will be labelled with [35S] L-methionine. The resulting proteins will be used for in vitro binding assays with GST-sorcin proteins. The latter will be either provided by unit of Colotti or will be produced in our laboratory. Bacterial lysates containing the GST-sorcin fusion proteins will be resuspended in PBS/20mM EDTA/1%Triton X-100, were incubated for 10 min at RT with the Glutathione-sepharose 4B beads to immobilise the fusion proteins. The beads will be washed with PBS containing 1% Triton X-100 and used for binding experiments. 10 µl of TNT protein extract will be incubated with the fusion proteins in PBS containing 1% Triton X-100, 1% BSA for 4 h at 4°C. The beads will be washed with PBS containing 1% Triton X-100 and bound proteins will be eluted and analysed by SDS-PAGE and autoradiography or by western blot.

b) Immunoprecipitation experiments: In vitro GST-pull down experiments will be extended and/or confirmed by the use of immunoprecipitation assays. For this purpose, NIH3T3 cells will be transfected with the same plasmids used in a) together with plasmids coding for sorcin (either cloned in frame with a HA tag or untagged). Transfected cells will be harvested and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 5 µg/ml Aprotinin, 5 µg/ml Leupeptin, 1% Triton x-100, 1% Sodium deoxycholate). Protein lysate will be pre-clear by adding 2 µL of mouse or rabbit serum (depending on the primari antibody that will be used) and incubated in a rotator for 30 minutes at room temperature. Sorcin-RyR2 complexes will be immunoprecipitated by adding the primary antibody to 500 mg of protien lysate and incubated overnight at 4° C. After the incubation, 50-100 µl of protein A conjugated-agarose beads will be added and incubated for 15-60 min. at 4 °C by gently mixing the sample with a shaker. Immunoprecipitated complexes will be collected by centrifugation and loaded on a SDS-PAGE gel for analysis.

Once identified the region in RyR2 responsible for sorcin interaction a more detailed characterization will be perfomed together with unit Colotti, who, among other approaches, will make use of plasmon resonance (SPR) analysis to study the specific interaction between defined regions of RyR2, allowing both kinetic and affinity analysis. It will be interesting to verify whether the region of interaction with sorcin in RyR2 may correspond to one of the four hot spots for RyR2 mutations linked to CPTV. In this case, the ability of sorcin to regulate mutated RyR2 channels will be also investigated.

Finally in view of the high degree of homology shared by the three RyR isoforms, upon identification of the region of RyR2 responsible for its interaction with sorcin, we will proceed with a sequence comparison with the corresponding regions of RyR1 and RyR3 isoforms. Should we identify a significant degree of homology we will extend our studies to the two other isoforms. Accordingly we will clone and express corresponding regions of RyR1 and RyR3 in order to evaluate their ability to bind sorcin, by in vitro pull-down and immunoprecipitation experiments.

CK2-dependent RyR2 phosphorylation:
RyR2 phosphorylation is regarded as an important regulatory mechanism of the channel activity. Although with controversial effects, phosphorylation of RyR2 has been shown to occur at Ser 2808 by PKA. In addition to Ser 2808, Ser 2030 has been identified as another major PKA phosphorylation site (Xiao et al., 2005), suggesting that PKA-mediated phosphorylation of other residues (including those that remain to be identified) may be important for RyR2 regulation. Finally, Ser 2808 is also a substrate for CaMKII phosphorylation. The role of other protein kinases on RyRs has not being investigated so far. Computer analysis of the RyR2 sequence has revealed the presence of several putative binding sites for protein kinases, including CK2. On this line, in collaboration with unit Ruzzene, we propose to verify weather RyR2 may be a target for CK2 phosphorylation, Microsomal proteins prepared from heart muscle, as well recombinant RyR2 channels and/or fragments of the cytoplasmic domain of RyR2 tested in collaboration with the Ruzzene unit to perform in vitro phosphorylation studies. Should CK2 phosphorylation of RyR2 be confirmed, we will proceed to characterize the functional properties of phosphorylated channels by means of in vitro [3H]-ryanodine binding experiments, and using CK2 inhibitors in in vitro cultured cardiomyocites.

a) [3H]-ryanodine binding: [3H]-ryanodine binding will be performed on SR cardiac membranes and or recombinant RyR2 channels expressed in HEK293 cells (60 µg) in incubation medium composed of 1 mM benzamidine, 1 mM PMSF, 0.5 µg/ml leupeptin, 1 µg/ml pepstatin A as protease inhibitors and 1 µM okadaic acid as phosphatase inhibitor, [Ca2+] in the range of 0.1 to 100 µM, 10 nM [3H]-ryanodine. Incubation will be performed for 90 min at 36 °C. Samples will be run in duplicate, filtered onto glass fiber filters and washed. The specific binding will be defined as the differente between the binding in the absence (total binding) and in the presence (non-specific binding) of 10 µM unlabeled ryanodine.

b) Calcium release properties of RyR2 channels will be studied in cardiomyocites from rat cultured in the presence or in the absence of CK2 inhibitors. Among other parameters, the sensibility to channel activators as well as the levels of store-overload-induced Ca2+ release (SOICR) will be evaluated by single cell analysis.

RyR2 regulation by sorcin:
Different studies have shown that sorcin can be phosphorylated by different protein kinases, including PKA and caMKII and that phosphorylation can affect the ability of sorcin to negatively modulate RyR2. Recently it has been observed that sorcin can interact with CK2 and from a sequence analysis, it can be postulated that sorcin can represent a possibile substrate of CK2, as itcontains residues in the correct consensus sequence for this kinase (Pinna and Ruzzene, 1996). Based on this observation, it will be interesting to verify whether and how sorcin phosphrylation can affect its ability to regulate RyR2. To this aim, in collaboration with unit Ruzzene, we are planning to perform ryanodine binding experiments on RyR2 channels. Microsomes will be prepared from heart muscle and used for binding assays in the presence of different concentrations of non-phosphorylated or CK2-phosphorylated sorcin. Experiments will be performed under free Ca2+ concentrations allowing for minimal to maximal channel opening.