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Homer Regulates Gain of Ryanodine Receptor Type 1 Channel Complex

Homer Regulates Gain of Ryanodine Receptor Type 1 Channel Complex THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 47, Issue of November 22, pp. 44722–44730, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Homer Regulates Gain of Ryanodine Receptor Type 1 Channel Complex* Received for publication, July 30, 2002, and in revised form, August 27, 2002 Published, JBC Papers in Press, September 9, 2002, DOI 10.1074/jbc.M207675200 ¶ ¶ Wei Feng‡, Jiancheng Tu§, Tianzhong Yang , Patty Shih Vernon§, Paul D. Allen , Paul F. Worley§, and Isaac N. Pessah‡ From the ‡Department of Molecular Biosciences, University of California, Davis, California 95616, the §Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, and the Department of Anesthesia, Preoperative and Pain Medicine, Brigham and Women’s Hospital, Boston, Massachusetts 02115 receptors (IP R) localized within endoplasmic and sarcoplasmic Homer proteins form an adapter system that regulates coupling of group 1 metabotropic glutamate receptors reticulum (SR) membranes (1), and cytoplasmic Shank proteins with intracellular inositol trisphosphate receptors and that are part of the N-methyl-D-aspartate receptor-associated is modified by neuronal activity. Here, we demonstrate PSD-95 complex (8, 9). A C-terminal coiled-coil domain is re- that Homer proteins also physically associate with ry- sponsible for Homer self-multimerization (10 –13). Although anodine receptors type 1 (RyR1) and regulate gating full-length Homer proteins are constitutively expressed in a responses to Ca , depolarization, and caffeine. In number of tissues, immediate-early gene products of the Hom- contrast to the prevailing notion of Homer function, er1 gene including Homer1a and Ania 3 lack the CC domain (3, Homer1c (long form) and Homer1-EVH1 (short form) 10). “Short form” Homer1a is rapidly and transiently induced evoke similar changes in RyR activity. The EVH1 do- by physiological synaptic stimuli that evoke long term poten- main mediates these actions of Homer and is selectively tiation in the hippocampus (3, 10) or in striatum by the addi- blocked by a peptide that mimics the Homer ligand. 1B5 tion of dopaminergic drugs (3). Thus, a use-dependent ex- dyspedic myotubes expressing RyR1 with a point muta- change of multimeric and protomeric short forms of Homer tion of a putative Homer-binding domain exhibit signif- appears to be responsible for dynamic regulation of context-de- icantly reduced (33%) amplitude in their responses to pendent signaling in neurons. K depolarization compared with cells expressing wild The functional influences of Homer adaptors on targeted type protein. These results reveal that in addition to its protein have not been identified. Multi-PDZ domain proteins known role as an adapter protein, Homer is a direct have been reported to cluster membrane ion channels with the modulator of Ca release gain. Homer is the first exam- result that the channel become active, but this effect is mim- ple of an “adapter” that also modifies signaling proper- icked by agents that otherwise cross-link the channel (14). ties of its target protein. The present work reveals a Homer binding to mGluR1 was recently reported to define novel mechanism by which Homer directly modulates the function of its target protein RyR1 and excitation- agonist-independent activity of the receptor (15); however, this contraction coupling in skeletal myotubes. This form of report did not define a molecular mechanism. The direct con- regulation may be important in other cell types that sequence of forming Homer-IP R complexes on the dynamics of express Homer and RyR1. endoplasmic reticulum/SR Ca release and its possible contri- bution to temporal and spatial aspects of Ca signals remain unclear. Homer proteins are adapters that physically bind and func- Recently all three Homer mRNAs have also been detected in tionally couple target proteins (1, 2). Homer1, Homer2, and striated muscle, and Homer protein has been identified within Homer3 are encoded by three mammalian genes whose expres- skeletal and cardiac muscle (4, 5). Moreover, putative Homer sion is dynamically regulated by cellular activity (2) and can ligand sequences are found within both the type 1 ryanodine attain high levels of protein in the nervous system where their receptor (RyR1) and the  -subunit of the dihydropyridine 1S functional regulation of excitatory signaling has been studied receptor (DHPR; CACNA1S) of skeletal muscle (7). RyR1 as- (3–5). A common element of structure of all Homer proteins is sembles as tetrameric structures within junctional regions of an N-terminal Enabled/Vasp homology domain (EVH1) essen- SR where it forms large organized arrays (16). RyR1 forms tial for binding Homer ligands (6 –7). Crystallographic analysis physical associations with  -DHPR that are essential for 1S of the EVH1 domain binding surface has revealed its specific engaging reciprocal signaling units that are essential for exci- association with polyproline ligands (7) that have previously tation-contraction (E-C) coupling, a process whereby depolar- been defined within plasmalemmal glutamate receptors includ- ization in the T-tubules triggers Ca release from SR result- ing mGluR1a and mGluR5a/b (1), inositol 1,4,5-trisphosphate ing in muscle contraction (17). Although Homer ligand consensus sequences reside in RyR1, experimental evidence confirming the physical association of Homer protein with * This work was supported by National Institutes of Health Grants RyR1, its functional influence on channel gating, and its pos- AR17605 (to P. D. A. and I. N. P.), ES10173 and ES11269 (to I. N. P.), and DA10309 and MH01153 (to P. F. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This mate receptor; IP R, inositol 1,4,5-trisphosphate receptor(s); SR, sarco- article must therefore be hereby marked “advertisement” in accordance plasmic reticulum; Ry, ryanodine; RyR1, type 1 ryanodine receptor; with 18 U.S.C. Section 1734 solely to indicate this fact. DHPR, dihydropyridine receptor; E-C, excitation-contraction; GST, glu- To whom correspondence should be addressed: Dept. of Molecular tathione S-transferase; PBS, phosphate-buffered saline; MOPS, 4-mor- Biosciences, School of Veterinary Medicine, One Shields Ave., Univer- pholinepropanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethyl- sity of California, Davis, CA 95616. E-mail: [email protected]. ammonio]-1-propanesulfonic acid; BLM, bilayer lipid membrane; CICR, 1 2 2 The abbreviations used are: mGluR, group 1 metabotropic gluta- Ca -induced Ca release; HLM, Homer ligand mutation. 44722 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Homer Regulates Gain of RyR1 44723 sible physiological involvement in regulating E-C coupling are lacking. The presence of putative Homer ligand sequences on both RyR1 and  -DHPR within skeletal myotubes provides 1S an excellent system not only to understand the determinants underlying the interaction of Homer proteins with RyR but also to define their functional and physiological significance. The present paper shows that the direct physical interaction of Homer proteins with RyR1 results in a potent (nanomolar) amplification of channel responses to physiological and phar- macological signals. Homer-RyR1 complexes described here regulate the gain associated with E-C coupling and may likely have broad significance in physical and functional coupling of Ca release units in excitable cells. MATERIALS AND METHODS Preparation of SR Membranes—Junctional SR membrane vesicles enriched in RyR1 were prepared from skeletal muscle of New Zealand White rabbits according to the method of Saito et al. (18) with some modifications (19). The preparations were stored in 10% sucrose, 10 mM HEPES, pH 7.4, at 80 °C until needed. Functional experiments re- ported were performed on four different SR preparations. Expression and Purification of Homer Construct—GST fusion con- structs were made by PCR amplifying the H1c open reading frame and the N-terminal 360-bp fragment with in frame primers with SalI and NotI sites and inserting the PCR products into pGEX4T-2 (Pharmacia Corp.). Homer1-EVH1 W24A mutant was made with the Quik- TM Change site-directed mutagenesis kit (Stratagene). Mena EVH1 GST was a gift from Dr. Leahy (Johns Hopkins and Howard Hughes Medical Institute). GST fusion plasmids of H1c, Homer1-EVH1 W24A, and Mena were transformed into BL21 cells, and positive clones were ex- panded. The cells were lysed by sonication, and the lysate was added to FIG.1. Physical association of homer with RyR. The physical 1 ml of glutathione-agarose (Sigma) and sequentially washed as de- interactions between RyR1 and Homer proteins were analyzed by co- scribed previously (8, 9). A slurry of glutathione-agarose beads loaded immunoprecipitation (a), GST-pull-down assays (b), and Western anal- with fusion protein was incubated with 5 units of biotinylated thrombin ysis of purified junctional SR membranes (c). In a, extracts of skeletal (Novagen). Purified Homer proteins were dialyzed against phosphate- muscle SR in 1% CHAPS buffer were incubated with Homer antibodies buffered saline (PBS) at 4 °C overnight. (Homer1c, Homer2, and Homer3). The immunoprecipitates were Construction and Expression of Putative Homer-binding Mutations probed with anti-RyR antibody 34C (22). Control lanes (lanes denoted in RyR1 cDNA—A 0.5-kb fragment was isolated between nucleotide as H1 PI, H2 PI, and H3 PI) utilized the preimmune serum of each 5064 (Pml site) and nucleotide 5582 (BglII site), and one 0.9-kb frag- antibody. The last lane (6 Lysate) was the offered sample. In b, pull- ment (between nucleotide 14312, ClaI site and 15230, XbaI site) was down assays with GST-Homer fusion proteins and detergent extracts of isolated from the full-length RyR1 cDNA in pHSV/RyR1 to make the skeletal muscle enriched in RyR1 were performed. RyR1 bound F1777R and F1782R mutations, respectively. The fragments were Homer1c and Homer3 GST proteins, whereas they did not bind to GST TM alone. In c, junctional SR membranes from four rabbits were separately cloned in pBluescript-SK(), and a QuikChange site-directed mu- prepared (preparations (preps)I–IV) and probed for RyR1 and Homer tagenesis kit (Stratagene) was used to introduce F1777R/F1782R into proteins by Western blot analysis. Junctional SR (30 g of protein from the fragments. After confirming their sequence, the mutated fragments each preparation) was applied onto SDS-PAGE, transferred to polyvi- were cloned into the full-length RyR1. Wild type and mutated cDNAs nylidene difluoride membrane, and probed by anti-RyR1, Homer1c, and were packaged in HSV-1 amplicon virions and expressed in 1B5 (RyR Homer3 antibodies. null) myotubes as previously described (21). Homer-binding Peptides—Peptide mGluR5WT, Pept (ALTPPSP- wt FRD), and mutant, Pept (ALTPPSPRRD) were synthesized on a PE before incubated with Cy-3-conjugated goat anti-mouse IgG or goat mut Biosystems 430A peptide synthesizer using N-(9-fluorenyl)methoxycar- anti-rabbit (1:1000) (Jackson ImmunoResearch Laboratories, Inc., West bonyl (Fmoc) chemistry, purified by reverse-phase high pressure liquid Grove, PA). Cy-3 fluorescence was visualized using a Nikon Diaphot chromatography, and analyzed by mass spectrometry. microscope (Nikon, Melville, NY) with an epifluorescence attachment Co-immunoprecipitation and GST Pull-down of Homer-RyR1 Com- (excitation, 510 –560 nm; emission, 590 nm). plex—Rat skeletal muscle was homogenized and sonicated in 20 vol- Gel electrophoresis and Western blot analysis were described in umes of ice-cold 10 mM MOPS buffer containing 1% CHAPS with detail elsewhere (23). Denatured protein (0.5–20 g) was loaded onto protease inhibitors and centrifuged for 10 min at 100,000  g 4 °C. 5% SDS-PAGE gels, electrophoresed, and then transferred onto polyvi- Three microliters of anti-Homer immune serum (8) or preimmune se- nylidene difluoride membranes. The blots were probed with 34C (1:200 rum was added to 60 l of tissue extract and incubated overnight at dilution) or anti-H1 polyclonal (1:100). The immunoblots were rinsed 4 °C. 50 l of protein A-Sepharose slurry was washed, added to the and then incubated either with a horseradish peroxidase-conjugated antibody-extract mixture, and incubated for an additional 2 h. The sheep anti-mouse IgG 1:20,000 (for blots probed with 34C) or goat beads were washed three times with ice-cold MOPS/CHAPS. Bound anti-rabbit IgG 1:10,000 (for blots probed with anti-H1; Sigma). En- protein was eluted with 4% SDS loading buffer and analyzed by SDS- hanced chemiluminescence techniques (PerkinElmer Life Sciences) PAGE and Western blot. For pull-down assays skeletal muscle deter- were used to visualize the immunoblots. gent extracts were prepared as described for co-immunoprecipitation Measurement of [ H]Ryanodine Binding—Equilibrium measurement 3 3 and mixed with glutathione beads loaded with GST or GST-Homer of specific high affinity [ H]ryanodine ([ H]Ry) binding was determined proteins, incubated overnight, and washed three times with ice-cold according to the method of Pessah et al. (24 –26). SR vesicles (50 g MOPS/CHAPS. protein/ml) were incubated with or without Homer protein (50 nM)in Immunocytochemical and Western Blot Analyses of Homer and RyR assay buffer containing 20 mM HEPES, pH 7.1, 250 mM KCl, 15 mM Proteins—Differentiated 1B5 myotubes were fixed in cold (20 °C) NaCl, 100 nM to 10 M CaCl , and 0.5 nM or5nM [ H]Ry for3hat37 °C. methanol and washed with PBS (Invitrogen). The cells were then per- Complete saturation binding curves were performed using the “cold meabilized before blocking with PBS with 5% normal goat serum. titration” method (24). The reactions were quenched by filtration Either anti-RyR1 34C monoclonal antibody (Developmental Studies through GF/B glass fiber filters and washed twice with ice-cold harvest Hybridoma Bank, University of Iowa, Iowa City, IA) (22) or anti-Hom- buffer (20 mM Tris-HCl, 250 mM KCl, 15 mM NaCl, 50 M CaCl ,pH er1 polyclonal were applied at concentrations of 1:25 and 1:10, respec- 7.1). Nonspecific binding was determined by incubating SR vesicles tively. The cells were washed with PBS with 5% normal goat serum with 1000-fold excess unlabelled ryanodine. 44724 Homer Regulates Gain of RyR1 FIG.2. Functional modulation of RyR1 by Homer. a and b show representative current traces of RyR1 channel activity in BLM before and after introduction of exogenous H1c (a) or H1-EVH1 (b) into the cis side of the channel. The current fluctuation through each channel was recorded at 40 mV. Cytosolic [Ca ] was 7 M. The open probability (P ) of the channel before and after exposed to the indicated concentration of Homer was averaged from a 2–5-min recording and denoted in the figure. The dashed lines indicate the open channel state, and the closed state (zero current) is indicated by the arrows (4C). The experiments were repeated with different single channels on separated BLM yielding similar results (a, n  4; b, n  3). c revealed that the binding of 5 nM (saturating) or 0.5 nM [ H]Ry to 50 g/ml of junctional SR-bound RyR1 in the presence of 350 nM free Ca was unaffected by control medium (CTRL), 50 nM GST fusion protein (GST), or a murine Ena homolog, Mena EVH1 (MENA). Either 50 nM Homer1c (H1c)or50nM Homer1c-GST (GST-H1c) enhanced occupancy 5-fold, whereas the Homer1 EVH1 (H1-EVH1) was less active (2-fold enhancement, p  0.05) when tested at saturating at 5 nM [ H]ryanodine. The H1-EVH1 point mutant at 50 nM (H1-W24A) lacked significant RyR1 activity. The data shown are the means  S.E. from at least four replicates. The maximum efficacy of 50 nM GST-H1c was 8-fold, and that of 150 nM H1-EVH1 was 5-fold when tested at 0.5 nM [ H]ryanodine (a concentration of ryanodine below its K ) showing that H1a-EVH1 3 2 is 3-fold less potent than H1c for enhancing [ H]ryanodine binding. d and e show that Homer enhances responsiveness of RyR1 to Ca and caffeine, respectively. SR vesicles (50 g/ml) were incubated as described under “Materials and Methods” for measuring macroscopic Ca fluxes. 2 2 After 160 M Ca was actively loaded into SR vesicles, 8 nM H1c (II) or the same volume of PBS (I) was introduced. After about 5 min, 80 M Ca 2 2 2 2 (d;Ca -induced Ca release) or 10 mM caffeine (e; caffeine-induced Ca release) was added to initiate release of Ca accumulated during the 2 2 loading phase. The initial Ca release rates in the presence (II) or absence (I) of H1c were analyzed and plotted. The initial rate of Ca -induced 2 1 1 2 Ca release increased from 0.002  0.03 to 0.337  0.029 mol mg min in the presence of H1c (n  16). Caffeine-induced Ca release 1 1 increased from 0.009  0.045 to 0.249  0.076 mol mg min in the presence of H1c (n  11). Single Channel Kinetics in Bilayer Lipid Membranes (BLM)—RyR1 was previously shown to accumulate into SR and support active Ca channels reconstitution was performed as described previously (19). loading without forming precipitates (30); however, the presence of The reconstituted channels exhibiting “low P type” gating behavior pyrophosphate buffers extravesicular Ca 10-fold, so that in its pres- 2 th 2 were the focus of the study. RyR1 is extremely sensitive to Cys oxida- ence the increase in free Ca is 1/10 of the total Ca added. tion during preparation of SR (19, 27) and results in 60 –70% of recon- Calcium Imaging of 1B5 Myotubes—The cultured and differentiated stitutions in BLM yielding channels that exhibit “high P type” gating o 1B5 (RyR null) myotubes were transduced with either wild type or behavior, whereas a small fraction of RyR1 channel reconstituted from F1777R/F1782R RyR1 amplicons as previously described (21). Within these preparations exhibit low P type gating behavior (19, 27–29). Low 24 –36 h after transduction, the cells were loaded with the calcium P type channels maintain low open probability over a large range of cis indicator dye Fluo-4 at 37 °C, for 30 min in imaging buffer (125 mM (cytoplasmic) Ca . Because intact muscle possesses a highly reducing NaCl, 5 mM KCl, 2 mM CaCl , 1.2 mM MgSO ,6mM glucose, and 25 mM 2 4 environment, the functional consequences of adding Homer proteins to HEPES, pH 7.4) supplemented with 0.05% bovine serum albumin and the cis chamber focused on reconstituted channels exhibiting low P 5 M Fluo-4 (Molecular Probes Inc., Eugene, OR). The cells were type gating behavior to better reflect the native environment within washed with imaging buffer before being transferred to a Nikon Dia- junctions of intact skeletal muscle (19, 27). Single channel activity was phot microscope. Fluo-4 was excited at 494 nm using the output of a measured at 40 mV (applied cis relative to the trans) using a patch DeltaRam fluorescence imaging system (Photon Technology Interna- clamp amplifier (Dagan 3900). The data were filtered at 1 kHz before tional, Princeton, NJ). Fluorescence emission was measured at 516 nm acquired at 10 kHz by a DigiData 1200A (Axon Inst., Foster City, CA). using a 40 quartz objective. Data presented as the 516 nm emissions The data were analyzed using pClamp 6 (Axon) without additional of Fluo-4 were collected with an intensified CCD camera (Solamere filtering. Average P was calculated from 1 min of recording. 2 2 Technology Group) from individual cells. Caffeine- and K -evoked Ca Macroscopic Ca Flux Measurement—Ca transport across SR transients were generated by exposing the cells for 10 s to increasing membranes was performed using the absorbance dye antipyrylazo III concentrations of caffeine or KCl. Each test was followed by a 30-s wash (30). SR membranes (50 g/ml) were equilibrated at 37 °C in a trans- allowing the intracellular Ca to return to base line. The data were port buffer consisting of 92 mM KCl, 20 mM K-MOPS (pH 7.0), 7.5 mM presented as 1) the percentages of myotubes responding to a given pyrophosphate, and 250 M antipyrylazo III. MgATP (1 mM), 10 g/ml stimulus and 2) the normalized magnitude of the transient, F  F /F creatine phosphokinase, and 5 mM phosphocreatine were present to o o (i.e. F/F ), where F is the mean base-line fluorescence at rest and F is regenerate ATP. Ca flux was monitored by measuring antipyrylazo o o III 710 –790 nm absorbance at 2– 4-s intervals using a diode array the peak fluorescence in response to the stimulus. The data were spectrophotometer (model 8452A, Hewlett Packard). Pyrophosphate recorded from at least 30 myotubes/treatment. Homer Regulates Gain of RyR1 44725 RESULTS AND DISCUSSION Homer Proteins Physically Interact with RyR1—An N-termi- nal EVH1 domain mediates Homer binding to target proteins by contacting specific proline-rich (Homer ligand) sequences found within mGluR1, mGluR5, and the IP R (7). Putative Homer ligand sequences are also present in RyR1 (7). Because Homer proteins are expressed in skeletal muscle (2), we exam- ined the possible in vivo association of Homer with RyR in immunoprecipitation assays from detergent (1% CHAPS) ex- tracts of rat skeletal muscle. Antibodies for Homer1c (H1c), Homer2, and Homer3 co-precipitated RyR1, whereas the pre- immune sera failed to do so (Fig. 1a). The interaction of RyR1 with Homer proteins was independently confirmed using GST pull-down assays with GST-Homer1c and GST-Homer3 fusion protein and detergent extracts of skeletal muscle (RyR1) (Fig. 1b; lanes GST-H1c and GST-H3). To examine the presence of endogenous Homer proteins in commonly used preparations of junctional SR enriched in RyR1, we isolated several membrane preparations from rabbit skeletal muscle by sucrose density gradient centrifugation and probed them by Western blot anal- ysis with RyR1- and Homer-specific antibodies (Fig. 1c). In every junctional SR preparation tested, H1c co-purified with RyR1, although the relative amounts of the H1c varied from preparation to preparation. Homer3 was also co-purified with RyR1 but was not detected in every junctional SR preparation examined (Fig. 1c). In all four preparations that were analyzed, H1c and Homer3 proteins were less enriched than RyR1. Be- cause Homer is a cytosolic protein, these results are consistent with the physical association of Homer with RyR1 and indicate that the co-enrichment of Homer relative to RyR1 varies from preparation to preparation. Presumably, Homer proteins vari- ably dissociated from the RyR1 complex during purification of junctional SR membranes. H1C and H1-EVH1 Enhance RyR1 Gating—To examine the functional consequence of the protein-protein interaction, we determined whether Homer could modify RyR1 channel gating behavior using the BLM method. Single RyR1 channels were reconstituted with an artificial BLM by inducing fusion of purified junctional SR vesicles. Those channels exhibiting the low P gating mode (19, 27–29) were selected for examining the influence of exogenous Homer protein added to the cytoplasmic (cis) side of the channel measured under voltage clamp condi- tions. Both the long form H1c (Fig. 2a) and short form H1- EVH1 lacking the coiled-coil domain (Fig. 2b) were highly potent and efficacious toward enhancing RyR1 channel open probability (P ). With 5 nM H1c added to the cis chamber, P o o increased 10-fold. H1-EVH1 was equally effective but 7-fold less potent (34 nM H1-EVH1 needed to enhance P 10-fold). The murine homolog of Ena (Mena) possesses an EVH1 domain that is structurally similar to Homer but binds a distinct pro- line-rich sequence (7). Mena lacked detectable activity on chan- nel P at the highest concentration tested (50 nM). Both H1- EVH1 and H1c shortened channel closed dwell time by over 100-fold without significantly altering open dwell time (data not shown). We also examined the effects of the immediate- early gene product Homer1a-EVH1 on high P gating mode channels reconstituted in BLM. Homer1a-EVH1 (40 nM) signif- chard analysis reveals that H1c increases maximum receptor occupancy (B ) 3-fold without altering affinity (K )(a and b; data points are the max D means  S.D. from two independent experiments each performed in FIG.3. Homer increases the maximum density of high affinity triplicate). c, H1c enhances the efficacy of Ca toward activating high 3 3 [ H]ryanodine binding and amplifies to junctional SR. a, equilib- affinity [ H]Ry binding 2.5-fold compared with control, without altering rium binding curves of [ H]ryanodine and 50 g/ml junctional SR were the half-activation constant (EC  210 versus 280 nM, respectively; performed using competition with unlabeled ryanodine (cold titration). mean of n  8). Free Ca concentration was adjusted by the addition The presence of 50 nM GST-H1c (H1C) increased occupancy over the of CaCl and EGTA based on calculations from Bound and Determined entire dose-response curve compared with the GST control. b, Scat- software (53). 44726 Homer Regulates Gain of RyR1 biochemical indicator of modulation of the open probability of the channel by exogenously added ligands (24 –26). When GST was included in the binding medium, occupancy of [ H]ryano- dine was indistinguishable from control (Fig. 2c). Similarly, [ H]ryanodine binding was unaltered by inclusion of Mena (100 nM). In marked contrast, 50 nM H1c or its fusion protein, GST-H1c, enhanced the binding of 5 nM [ H]ryanodine by nearly 5-fold (Fig. 2c). The actions of GST-H1c measured in the presence [ H]ryanodine at a concentration well below its K (0.5 nM) resulted in 8-fold enhancement of occupancy (Fig. 2c, right panel) consistent with the effects of Homer protein on enhancing channel P in BLM studies (Fig. 2a). At a concen- tration of 5 nM [ H]ryanodine, the addition of 50 nM H1-EVH1 mimicked the activity of full-length H1c but exhibited smaller (2-fold) but statistically significant (p  0.05) enhancement in binding compared with control. When tested at subsaturating [ H]ryanodine (0.5 nM), the addition of 150 nM H1-EVH1 in- creased binding 5-fold, whereas 50 nM H1c increased binding 8-fold (Fig. 2c). This demonstrates that both forms have a similar ability to enhance RyR1 activity consistent with their ability to enhance the P of channels reconstituted in BLM (Fig. 2b). The 3-fold difference in the potency observed with the long and short forms of Homer on RyR1 could be attributed to the multivalency of H1c that may optimize conformational interaction with RyR1 oligomeric units. As a further control, we found that the W24A point mutant of H1-EVH1, which does not bind Homer ligands (7), had negligible activity on altering the binding of [ H]ryanodine to RyR1 under the stated assay condi- tion (Fig. 2c). These results support the hypothesis that specific protein-protein interactions between Homer proteins and RyR1 can variably enhance the gain of signaling events mediated 2 2 2 through Ca -induced Ca release (CICR), and hence Ca released from SR, by influencing the fraction of RyR channels within junctional SR responsive to physiological activators. To further test the hypothesis, [ H]ryanodine-binding curves were performed in the presence of nearly optimal Ca for channel activation (1 M) and in the absence or presence of Homer protein (Fig. 3a). Scatchard analysis revealed that H1c enhanced the binding of [ H]ryanodine over the complete range of concentrations, enhancing maximum occupancy 3-fold (from B  4.2 and 12.7 pmol mg in the absence and presence of max 50 nM H1c, respectively) without significantly altering binding affinity (K  7.95 and 9.06 nM, respectively; Fig. 3b). These results indicate that formation of H1c-RyR1 complexes in vitro significantly increased the density of Ca -responsive channels within the SR membrane. Because Homer protein appears to dissociate from SR preparations during biochemical enrich- FIG.4. Enhancement of RyR1 channel function by Homer is ment of RyR1, the ability of exogenous Homer to increase the specifically blocked by competing peptide. H1c was preincubated density of functional RyR1 able to bind ryanodine in a Ca - with 1000 synthetic wild type Homer-binding peptide (Pept ), syn- wt dependent manner likely results from formation of new pro- thetic peptide missing the critical phenylalanine (Pept ), or PBS at mut 37 °C for 10 min. After completion of preloading of SR with 160 M tein-protein complexes that may have been lost during prepa- Ca , Homer in the presence or absence of Pept or Pept was wt mut ration of junctional SR membranes. introduced. The final concentration of the H1c in the transport buffer Although in skeletal muscle CICR is not necessary to initiate was 20 nM (a)or8nM (b and c). 10 M ryanodine (Ry) or Ruthenium Red E-C coupling, nor is it necessary to engage retrograde signaling (RR) was added to activate or inhibit RyR1, whereas 0.5 M thapsigar- gin (TG) was introduced to inhibit SR Ca pumps. In b and c, the final (32), it is generally agreed that Ca is an important physio- 2 2 concentration of Ca used to induce Ca release was 60 M. Summary logical modulator of RyR1 in skeletal muscle. Several ligands data represent the mean of 21 (a),6(b), or 6 (c) repetitions of these have been shown to influence the sensitivity of RyR to activa- experiments. 2 3 tion by Ca (24, 26). Using [ H]ryanodine binding analysis near K for the radioligand, H1c (50 nM) did not significantly icantly enhanced the gating activity of in the presence of 7 M shift the sensitivity of RyR1 to activation by Ca relative to cis Ca (4-fold; not shown). control (EC  210 versus 280 nM Ca , respectively; Fig. 3c). Mechanism of Homer Modulation of RyR1—The manner in which Homer proteins enhances RyR1 activity was examined However, H1c did enhance maximal occupancy 2.5-fold, indi- cating an enhanced “gain” of RyR1 toward activation by Ca in more detail by measuring their influence on the binding of 3 3 [ H]ryanodine to skeletal muscle JSR. Because [ H]ryanodine consistent with an increase in the density of Ca -responsive RyR1 complexes. binds with high affinity to an open state of the RyR channel, occupancy of [ H]ryanodine-binding sites provides a convenient Homer Enhances CICR and Caffeine—A functional corre- Homer Regulates Gain of RyR1 44727 FIG.5. Expression of RyR1 possessing a mutation of a putative Homer-ligand sequence in 1B5 myotubes. a shows that 1B5 myotubes constitutively express immunoreactive Homer protein. The upper panel shows 1B5 cells expressing wt RyR1 immunostained with pan Homer antibody as described under “Materials and Methods” at 10 magnification. The same results were observed with 1B5 myotubes expressing RyR1 possessing point mutation F1777R/F1782R. The lower panel shows results from Western analysis of the whole membrane fraction obtained from 1B5 myotubes and rabbit skeletal muscle junctional SR probed with Homer1c-selective antibody. b, immunocytochemical analysis of 1B5 myotubes at 10 before (left panel) and after transduction with HSV-1 virions containing wild type (wt) RyR1 (middle panel) and full-length RyR1 containing a point mutation within the putative Homer ligand domain (HLM, right panel). 100 magnification inserts in the middle and right panels show that both wild type and HLM RyR1 have the characteristic punctate staining pattern of RyR1 when it is targeted properly to peripheral junctions. late to the results obtained from radioligand receptor binding siveness of RyR1 at the level of the single channel and under- analysis was undertaken to understand how H1c influences lies the increased “gain” of RyR1-mediated Ca efflux units 2 2 macroscopic Ca efflux elicited by either Ca (CICR) or within junctional SR (defined by two independent measures, 2 3 caffeine from actively loaded skeletal muscle SR vesicles (Fig. macroscopic Ca efflux (CICR) and [ H]ryanodine binding 4, b and c). This assay utilizes pyrophosphate to support density). These actions of Homer protein exhibited a high de- active loading of Ca into the vesicles (30). After calcium gree of molecular specificity and were mediated by their unique loading, the addition of H1c to a subthreshold concentration EVH1 domain. (8 nM) that did not directly initiate net Ca efflux from the RyR1 Homer-binding Domain Regulates Gain—Conforma- SR vesicles enhanced the initial rate of RyR1-mediated tional changes in IP R caused by IP binding and store deple- 3 3 Ca elicited by subsequent addition of either optimal 80 M tion was shown to provide a retrograde signal to opening of 2 2 2 total Ca (8.0 M free Ca ) or saturating caffeine (10 mM) store-operated Ca channels in the plasma membrane (33, 7.9- and 8.2-fold, respectively. Thus, H1c enhanced the max- 34), similar to the bi-directional signaling between RyR1 and imum efficacy of both Ca and caffeine for activation of  -DHPR in skeletal muscle that is essential for engaging E-C 1S RyR1. coupling (17). Moreover, evidence for conformational coupling Homer Modulation of RyR1 Is Blocked by Specific Peptide— between RyR and store-operated Ca channels has been re- The specificity of H1c for enhancing Ca release from SR was cently presented (35, 36). Thus, conformational coupling ap- examined by preincubating H1c with a synthetic “blocking” pears to be a fundamental mechanism involved in generating peptide that mimics the Homer ligand within the C terminus of localized Ca signals. Both RyR and  -DHPR possess dis- 1S mGluR-1 (1). Fig. 4a (trace I) demonstrates that addition of crete polyproline sequences that are putative Homer ligands. saturating (20 nM) H1c induced net Ca release from actively RyR1 contains a putative Homer-ligand domain between amino loaded SR vesicles. In the absence of added Homer, Ca was acids 1773 and 1783. A second putative Homer binding domain not released until the addition of either ryanodine (10 M; trace can be found in RyR1 (PPPGYA; amino acids 804 – 809), where III) or the SR Ca pump inhibitor thapsigargin (0.5 M; trace the essential hydrophobic phenylalanine is substituted with IV). The effect of H1c could be blocked by the addition of 50 M tyrosine. wild type peptide (Pept ) but not by a point mutant of the We therefore tested the hypothesis that Homer is involved in wt blocking peptide (Pept ) that lacks Homer binding activity modulating the strength of depolarized-induced Ca release mut (1) (Fig. 4a, trace II). Pept does not block release by subse- in intact cells. 1B5 dyspedic skeletal myotubes express all the wt quent addition of ryanodine. Pept , but not Pept , also pre- proteins known to be associated with the mature triadic junc- wt mut vented the ability of H1c to amplify CICR after active loading of tion but lack expression of the three RyR isoforms (23, 37). 160 M Ca into SR (Fig. 4, b and c). CICR induced by the Transduction of 1B5 myotubes with wild type RyR1 cDNA addition of 60 M Ca in the presence of 8 nM H1c exhibited reconstituted bi-directional signaling between RyR1 and  - 1S indistinguishable rates of Ca release in the presence or ab- DHPR and directed the organization of  -DHPR particles 1S sence of Pept (Fig. 4c, traces I and II). Subsequent addition into organized arrays of tetrads (38, 39). Homer1 protein was mut of Ruthenium Red (10 M), an RyR channel blocker, resulted in detected in 1B5 myotubes by immunocytochemical and West- active re-uptake of Ca into SR vesicles. However, in the ern blot analyses (Fig. 5a). presence of Pept (Fig. 4b, trace III), the addition of 60 M The possible functional role of one of the putative Homer- wt 2 2 2 Ca displayed a net Ca uptake into SR because SR Ca binding sites of RyR1 was tested by incorporating site-directed pump-transport into the vesicles surpassed the amount of re- mutations (F1777R/F1782R) within the full-length protomer. lease through RyR1. Active accumulation could be interrupted The substitutions were chosen based on the recent data show- abruptly and was quickly followed by Ca release after sub- ing that hydrophobic phenylalanine C-terminal to polyprolines sequent addition of 10 M ryanodine (Fig. 4, b and c, trace IV). are essential for Homer binding (7). RyR protomers from 1B5 As further evidence of its specificity, we confirmed that Pept myotubes expressing full-length wild type and F1777R/F1782R wt did not affect modulation of RyR1 by the scorpion toxin mau- RyR1 proteins exhibited the same mobility by SDS-PAGE (not rocalcine, a potent activator of RyR1 (31) (not shown). shown) and show similar levels of expression by immunocyto- Taken together, these results revealed that physical associ- chemistry. At higher magnification both wt and F1777R/ ation of H1c with RyR1 dramatically enhanced Ca respon- F1782R RyR1 exhibited the characteristic punctate pattern of 44728 Homer Regulates Gain of RyR1 FIG.6. Mutation of a putative Homer-ligand sequence of RyR1 attenuates excitation-contraction coupling in 1B5 myotubes. a shows the responses of 1B5 myotubes expressing wild type and the putative HLM (F1777R/F1782R RyR1) to threshold (1 mM) and saturating (30 mM) caffeine. 40% fewer myotubes responded to the threshold concentration of caffeine, whereas all myotubes responded to saturating caffeine. The normalized amplitude of responses to threshold caffeine were 56% smaller (p  0.001) in HLM versus wild type but were not different at saturating caffeine. The data were analyzed from 68 and 92 wild type and HLM myotubes, respectively. c and d show the K -evoked Ca transients following Homer Regulates Gain of RyR1 44729 expression within the myotubes, which has previously been The present findings reveal that Homer proteins through shown to be consistent with targeting of RyR protein to periph- their direct interaction with RyR1 can modulate the gain of E-C eral junctions (Fig. 5b) (38, 39). coupling. This can occur in three ways. First, the interaction of Both wild type and the putative Homer ligand mutation constitutively expressed H1c could bind to one to four channel (HLM; F1777R/F1782R) RyR1 responded to caffeine chal- protomers to regulate cooperativity associated with channel lenges. However, the percentage of HLM myotubes responding activation. Second, H1c multimers could cross-link multiple to a threshold concentration of caffeine (1 mM) was 39.6% lower RyR1 oligomers to facilitate coupled gating among channels than wild type myotubes (43.5% versus 72.0% of n  92 and 68 elicited by FKBP12 (40). Because localized Ca sparks occur myotubes, respectively; Fig. 6a, left bars). When challenged upon release of Ca through a cluster of RyR (41), it is inter- with saturating caffeine (30 mM), the frequency of responses esting to note that H1c has been recently shown to induce with HLM and wild type myotubes were both 100%. The nor- sparks in frog skeletal muscle (42). Homer-RyR associations malized magnitude of the Fluo-4 fluorescence signal (F/F )isa o may therefore be a significant physiological regulator of spark quantitative measure of the amount of Ca mobilized in re- activity that contributes to Ca waves that sweep through sponse to direct activation of release channels by caffeine. The cells and subsequently activate myocyte contraction. Third, magnitude of the responses to 1 mM caffeine was 56.6% smaller multimeric H1c could directly cross-link RyR1 with  -DHPR 1S (F/F  6.8% versus 15.7%; p  0.003) in myotubes expressing o to enhance the efficiency of E-C coupling in a manner already HLM. However, 30 mM caffeine elicited Ca responses whose described for mGluR1 and IP R (1, 7). Whether Homer inter- magnitude were indistinguishable between wild type and HLM actions with a putative binding site on  -DHPR contribute to 1S myotubes, indicating that the SR stores of both cell types were modulation of E-C coupling remains to be determined. filled with Ca to similar levels (Fig. 6a, right bars). Homer short form (i.e. H1a-EVH1) is encoded by a prema- Expression of either wild type or HLM cDNAs in 1B5 myo- turely terminated transcript that lacks the coiled-coil domain tubes also restored Ca responses to K depolarization con- that is required for the formation of Homer multimers. The sistent with restoration of skeletal type excitation-contraction currently favored mechanism for how Homer proteins regulate coupling, and the amplitude of this response increased in a signaling functions suggests that the H1-EVH1, an immediate- saturable manner with increasing K concentration between early gene product, is rapidly expressed in response to cellular 10 and 100 mM (Fig. 6, c and d). The expressed proteins pro- activity, whereas long form H1c is constitutively expressed. In duced Ca transients that were kinetically identical, and the this scheme, H1c plays an adapter function, linking target apparent potency for producing K -evoked Ca transients proteins such as mGluR1 and IP R in neurons. Immediate- was unaltered by the mutation (EC  26.6  1.2 and 27.0 early expression of H1-EVH1 acts in a dominate-negative fash- 1.2 mM for wild type and mutant protein, respectively). Impor- ion by competing with existing H1c complexes, thereby impair- tantly, however, the maximum amplitude of the K -evoked ing the formation and maintenance of Homer-mediated cluster Ca transients produced by cells expressing HLM were 33% of protein complexes (2). However, experimental evidence for a smaller than those produced by wild type RyR1 (mean F/F more dynamic role for Homer proteins in cellular regulation 76  0.85 and 58  2.0; n  34 and 48 cells for wild type and has recently been defined (13, 43– 45). For example, experi- HLM, respectively; p  0.001), demonstrating Homer’s impor- ments using single particle tracking demonstrated that the tance in modulating depolarization induced Ca release. The composition of Homer-mediated clustering of protein com- possible existence of a second Homer ligand domain within plexes is highly dynamic. The exchange of the proteins between RyR1 (amino acids 804 – 809) and its functional role remains to dispersed and clustered states is fast and is regulated during be investigated. physiological processes (44, 45). The present results suggest Functional Significance of Interactions between RyR1 and that the catalytic activity of Homer proteins toward RyR may Homer Proteins—The present study demonstrates that Homer provide one mechanism linking Ca signals with dynamic binding directly enhances responses of RyR1 to physiological regulation of Homer-mediated protein clustering. and pharmacological stimuli. This activity is conferred by the One anticipated consequence of Homer catalytic activity is to EVH1 domain and does not require the cross-linking adapter increase the spatial fidelity of signaling beyond what can be function of Homer. However, multimeric H1c does appear to achieved by simple physical coupling. Homer and RyR proteins significantly enhance the potency toward enhancing RyR1 re- are widely expressed, and it may be anticipated that their sponses. Homer is the first example of an “adapter” that also physical and functional interactions identified here contribute modifies signaling properties of its target protein. Multi-PDZ to temporal and spatial patterning of intracellular Ca signal- domain proteins are reported to activate ion channels, but this ing. In skeletal muscle, release of Ca through RyR1 is the key activity is mediated by simple physical cross-linking (14). The event linking membrane depolarization and mechanical activ- nanomolar affinities required for channel modulation with full- ity during E-C coupling. In neurons, Homer proteins play a length RyR and the complete long and short forms of Homer special role in adaptation to cellular activity. CICR has been indicate a structurally specific interaction. Previous studies demonstrated in a number of neuronal cell types (46 – 48). that used peptides to block the binding of EVH domains with Evidence for the role of RyR in the regulation of specific aspects their target proteins suggested affinities in the micromolar of neuronal plasticity, dendritic growth, and spatial learning range (1, 8). Because our studies used full-length proteins, it is has recently been demonstrated (49 –52). The present results inferred that additional structure is important for optimizing predict that RyR1-Homer interactions may also contribute el- these binding interactions. 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Homer Regulates Gain of Ryanodine Receptor Type 1 Channel Complex

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 47, Issue of November 22, pp. 44722–44730, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Homer Regulates Gain of Ryanodine Receptor Type 1 Channel Complex* Received for publication, July 30, 2002, and in revised form, August 27, 2002 Published, JBC Papers in Press, September 9, 2002, DOI 10.1074/jbc.M207675200 ¶ ¶ Wei Feng‡, Jiancheng Tu§, Tianzhong Yang , Patty Shih Vernon§, Paul D. Allen , Paul F. Worley§, and Isaac N. Pessah‡ From the ‡Department of Molecular Biosciences, University of California, Davis, California 95616, the §Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, and the Department of Anesthesia, Preoperative and Pain Medicine, Brigham and Women’s Hospital, Boston, Massachusetts 02115 receptors (IP R) localized within endoplasmic and sarcoplasmic Homer proteins form an adapter system that regulates coupling of group 1 metabotropic glutamate receptors reticulum (SR) membranes (1), and cytoplasmic Shank proteins with intracellular inositol trisphosphate receptors and that are part of the N-methyl-D-aspartate receptor-associated is modified by neuronal activity. Here, we demonstrate PSD-95 complex (8, 9). A C-terminal coiled-coil domain is re- that Homer proteins also physically associate with ry- sponsible for Homer self-multimerization (10 –13). Although anodine receptors type 1 (RyR1) and regulate gating full-length Homer proteins are constitutively expressed in a responses to Ca , depolarization, and caffeine. In number of tissues, immediate-early gene products of the Hom- contrast to the prevailing notion of Homer function, er1 gene including Homer1a and Ania 3 lack the CC domain (3, Homer1c (long form) and Homer1-EVH1 (short form) 10). “Short form” Homer1a is rapidly and transiently induced evoke similar changes in RyR activity. The EVH1 do- by physiological synaptic stimuli that evoke long term poten- main mediates these actions of Homer and is selectively tiation in the hippocampus (3, 10) or in striatum by the addi- blocked by a peptide that mimics the Homer ligand. 1B5 tion of dopaminergic drugs (3). Thus, a use-dependent ex- dyspedic myotubes expressing RyR1 with a point muta- change of multimeric and protomeric short forms of Homer tion of a putative Homer-binding domain exhibit signif- appears to be responsible for dynamic regulation of context-de- icantly reduced (33%) amplitude in their responses to pendent signaling in neurons. K depolarization compared with cells expressing wild The functional influences of Homer adaptors on targeted type protein. These results reveal that in addition to its protein have not been identified. Multi-PDZ domain proteins known role as an adapter protein, Homer is a direct have been reported to cluster membrane ion channels with the modulator of Ca release gain. Homer is the first exam- result that the channel become active, but this effect is mim- ple of an “adapter” that also modifies signaling proper- icked by agents that otherwise cross-link the channel (14). ties of its target protein. The present work reveals a Homer binding to mGluR1 was recently reported to define novel mechanism by which Homer directly modulates the function of its target protein RyR1 and excitation- agonist-independent activity of the receptor (15); however, this contraction coupling in skeletal myotubes. This form of report did not define a molecular mechanism. The direct con- regulation may be important in other cell types that sequence of forming Homer-IP R complexes on the dynamics of express Homer and RyR1. endoplasmic reticulum/SR Ca release and its possible contri- bution to temporal and spatial aspects of Ca signals remain unclear. Homer proteins are adapters that physically bind and func- Recently all three Homer mRNAs have also been detected in tionally couple target proteins (1, 2). Homer1, Homer2, and striated muscle, and Homer protein has been identified within Homer3 are encoded by three mammalian genes whose expres- skeletal and cardiac muscle (4, 5). Moreover, putative Homer sion is dynamically regulated by cellular activity (2) and can ligand sequences are found within both the type 1 ryanodine attain high levels of protein in the nervous system where their receptor (RyR1) and the  -subunit of the dihydropyridine 1S functional regulation of excitatory signaling has been studied receptor (DHPR; CACNA1S) of skeletal muscle (7). RyR1 as- (3–5). A common element of structure of all Homer proteins is sembles as tetrameric structures within junctional regions of an N-terminal Enabled/Vasp homology domain (EVH1) essen- SR where it forms large organized arrays (16). RyR1 forms tial for binding Homer ligands (6 –7). Crystallographic analysis physical associations with  -DHPR that are essential for 1S of the EVH1 domain binding surface has revealed its specific engaging reciprocal signaling units that are essential for exci- association with polyproline ligands (7) that have previously tation-contraction (E-C) coupling, a process whereby depolar- been defined within plasmalemmal glutamate receptors includ- ization in the T-tubules triggers Ca release from SR result- ing mGluR1a and mGluR5a/b (1), inositol 1,4,5-trisphosphate ing in muscle contraction (17). Although Homer ligand consensus sequences reside in RyR1, experimental evidence confirming the physical association of Homer protein with * This work was supported by National Institutes of Health Grants RyR1, its functional influence on channel gating, and its pos- AR17605 (to P. D. A. and I. N. P.), ES10173 and ES11269 (to I. N. P.), and DA10309 and MH01153 (to P. F. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This mate receptor; IP R, inositol 1,4,5-trisphosphate receptor(s); SR, sarco- article must therefore be hereby marked “advertisement” in accordance plasmic reticulum; Ry, ryanodine; RyR1, type 1 ryanodine receptor; with 18 U.S.C. Section 1734 solely to indicate this fact. DHPR, dihydropyridine receptor; E-C, excitation-contraction; GST, glu- To whom correspondence should be addressed: Dept. of Molecular tathione S-transferase; PBS, phosphate-buffered saline; MOPS, 4-mor- Biosciences, School of Veterinary Medicine, One Shields Ave., Univer- pholinepropanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethyl- sity of California, Davis, CA 95616. E-mail: [email protected]. ammonio]-1-propanesulfonic acid; BLM, bilayer lipid membrane; CICR, 1 2 2 The abbreviations used are: mGluR, group 1 metabotropic gluta- Ca -induced Ca release; HLM, Homer ligand mutation. 44722 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Homer Regulates Gain of RyR1 44723 sible physiological involvement in regulating E-C coupling are lacking. The presence of putative Homer ligand sequences on both RyR1 and  -DHPR within skeletal myotubes provides 1S an excellent system not only to understand the determinants underlying the interaction of Homer proteins with RyR but also to define their functional and physiological significance. The present paper shows that the direct physical interaction of Homer proteins with RyR1 results in a potent (nanomolar) amplification of channel responses to physiological and phar- macological signals. Homer-RyR1 complexes described here regulate the gain associated with E-C coupling and may likely have broad significance in physical and functional coupling of Ca release units in excitable cells. MATERIALS AND METHODS Preparation of SR Membranes—Junctional SR membrane vesicles enriched in RyR1 were prepared from skeletal muscle of New Zealand White rabbits according to the method of Saito et al. (18) with some modifications (19). The preparations were stored in 10% sucrose, 10 mM HEPES, pH 7.4, at 80 °C until needed. Functional experiments re- ported were performed on four different SR preparations. Expression and Purification of Homer Construct—GST fusion con- structs were made by PCR amplifying the H1c open reading frame and the N-terminal 360-bp fragment with in frame primers with SalI and NotI sites and inserting the PCR products into pGEX4T-2 (Pharmacia Corp.). Homer1-EVH1 W24A mutant was made with the Quik- TM Change site-directed mutagenesis kit (Stratagene). Mena EVH1 GST was a gift from Dr. Leahy (Johns Hopkins and Howard Hughes Medical Institute). GST fusion plasmids of H1c, Homer1-EVH1 W24A, and Mena were transformed into BL21 cells, and positive clones were ex- panded. The cells were lysed by sonication, and the lysate was added to FIG.1. Physical association of homer with RyR. The physical 1 ml of glutathione-agarose (Sigma) and sequentially washed as de- interactions between RyR1 and Homer proteins were analyzed by co- scribed previously (8, 9). A slurry of glutathione-agarose beads loaded immunoprecipitation (a), GST-pull-down assays (b), and Western anal- with fusion protein was incubated with 5 units of biotinylated thrombin ysis of purified junctional SR membranes (c). In a, extracts of skeletal (Novagen). Purified Homer proteins were dialyzed against phosphate- muscle SR in 1% CHAPS buffer were incubated with Homer antibodies buffered saline (PBS) at 4 °C overnight. (Homer1c, Homer2, and Homer3). The immunoprecipitates were Construction and Expression of Putative Homer-binding Mutations probed with anti-RyR antibody 34C (22). Control lanes (lanes denoted in RyR1 cDNA—A 0.5-kb fragment was isolated between nucleotide as H1 PI, H2 PI, and H3 PI) utilized the preimmune serum of each 5064 (Pml site) and nucleotide 5582 (BglII site), and one 0.9-kb frag- antibody. The last lane (6 Lysate) was the offered sample. In b, pull- ment (between nucleotide 14312, ClaI site and 15230, XbaI site) was down assays with GST-Homer fusion proteins and detergent extracts of isolated from the full-length RyR1 cDNA in pHSV/RyR1 to make the skeletal muscle enriched in RyR1 were performed. RyR1 bound F1777R and F1782R mutations, respectively. The fragments were Homer1c and Homer3 GST proteins, whereas they did not bind to GST TM alone. In c, junctional SR membranes from four rabbits were separately cloned in pBluescript-SK(), and a QuikChange site-directed mu- prepared (preparations (preps)I–IV) and probed for RyR1 and Homer tagenesis kit (Stratagene) was used to introduce F1777R/F1782R into proteins by Western blot analysis. Junctional SR (30 g of protein from the fragments. After confirming their sequence, the mutated fragments each preparation) was applied onto SDS-PAGE, transferred to polyvi- were cloned into the full-length RyR1. Wild type and mutated cDNAs nylidene difluoride membrane, and probed by anti-RyR1, Homer1c, and were packaged in HSV-1 amplicon virions and expressed in 1B5 (RyR Homer3 antibodies. null) myotubes as previously described (21). Homer-binding Peptides—Peptide mGluR5WT, Pept (ALTPPSP- wt FRD), and mutant, Pept (ALTPPSPRRD) were synthesized on a PE before incubated with Cy-3-conjugated goat anti-mouse IgG or goat mut Biosystems 430A peptide synthesizer using N-(9-fluorenyl)methoxycar- anti-rabbit (1:1000) (Jackson ImmunoResearch Laboratories, Inc., West bonyl (Fmoc) chemistry, purified by reverse-phase high pressure liquid Grove, PA). Cy-3 fluorescence was visualized using a Nikon Diaphot chromatography, and analyzed by mass spectrometry. microscope (Nikon, Melville, NY) with an epifluorescence attachment Co-immunoprecipitation and GST Pull-down of Homer-RyR1 Com- (excitation, 510 –560 nm; emission, 590 nm). plex—Rat skeletal muscle was homogenized and sonicated in 20 vol- Gel electrophoresis and Western blot analysis were described in umes of ice-cold 10 mM MOPS buffer containing 1% CHAPS with detail elsewhere (23). Denatured protein (0.5–20 g) was loaded onto protease inhibitors and centrifuged for 10 min at 100,000  g 4 °C. 5% SDS-PAGE gels, electrophoresed, and then transferred onto polyvi- Three microliters of anti-Homer immune serum (8) or preimmune se- nylidene difluoride membranes. The blots were probed with 34C (1:200 rum was added to 60 l of tissue extract and incubated overnight at dilution) or anti-H1 polyclonal (1:100). The immunoblots were rinsed 4 °C. 50 l of protein A-Sepharose slurry was washed, added to the and then incubated either with a horseradish peroxidase-conjugated antibody-extract mixture, and incubated for an additional 2 h. The sheep anti-mouse IgG 1:20,000 (for blots probed with 34C) or goat beads were washed three times with ice-cold MOPS/CHAPS. Bound anti-rabbit IgG 1:10,000 (for blots probed with anti-H1; Sigma). En- protein was eluted with 4% SDS loading buffer and analyzed by SDS- hanced chemiluminescence techniques (PerkinElmer Life Sciences) PAGE and Western blot. For pull-down assays skeletal muscle deter- were used to visualize the immunoblots. gent extracts were prepared as described for co-immunoprecipitation Measurement of [ H]Ryanodine Binding—Equilibrium measurement 3 3 and mixed with glutathione beads loaded with GST or GST-Homer of specific high affinity [ H]ryanodine ([ H]Ry) binding was determined proteins, incubated overnight, and washed three times with ice-cold according to the method of Pessah et al. (24 –26). SR vesicles (50 g MOPS/CHAPS. protein/ml) were incubated with or without Homer protein (50 nM)in Immunocytochemical and Western Blot Analyses of Homer and RyR assay buffer containing 20 mM HEPES, pH 7.1, 250 mM KCl, 15 mM Proteins—Differentiated 1B5 myotubes were fixed in cold (20 °C) NaCl, 100 nM to 10 M CaCl , and 0.5 nM or5nM [ H]Ry for3hat37 °C. methanol and washed with PBS (Invitrogen). The cells were then per- Complete saturation binding curves were performed using the “cold meabilized before blocking with PBS with 5% normal goat serum. titration” method (24). The reactions were quenched by filtration Either anti-RyR1 34C monoclonal antibody (Developmental Studies through GF/B glass fiber filters and washed twice with ice-cold harvest Hybridoma Bank, University of Iowa, Iowa City, IA) (22) or anti-Hom- buffer (20 mM Tris-HCl, 250 mM KCl, 15 mM NaCl, 50 M CaCl ,pH er1 polyclonal were applied at concentrations of 1:25 and 1:10, respec- 7.1). Nonspecific binding was determined by incubating SR vesicles tively. The cells were washed with PBS with 5% normal goat serum with 1000-fold excess unlabelled ryanodine. 44724 Homer Regulates Gain of RyR1 FIG.2. Functional modulation of RyR1 by Homer. a and b show representative current traces of RyR1 channel activity in BLM before and after introduction of exogenous H1c (a) or H1-EVH1 (b) into the cis side of the channel. The current fluctuation through each channel was recorded at 40 mV. Cytosolic [Ca ] was 7 M. The open probability (P ) of the channel before and after exposed to the indicated concentration of Homer was averaged from a 2–5-min recording and denoted in the figure. The dashed lines indicate the open channel state, and the closed state (zero current) is indicated by the arrows (4C). The experiments were repeated with different single channels on separated BLM yielding similar results (a, n  4; b, n  3). c revealed that the binding of 5 nM (saturating) or 0.5 nM [ H]Ry to 50 g/ml of junctional SR-bound RyR1 in the presence of 350 nM free Ca was unaffected by control medium (CTRL), 50 nM GST fusion protein (GST), or a murine Ena homolog, Mena EVH1 (MENA). Either 50 nM Homer1c (H1c)or50nM Homer1c-GST (GST-H1c) enhanced occupancy 5-fold, whereas the Homer1 EVH1 (H1-EVH1) was less active (2-fold enhancement, p  0.05) when tested at saturating at 5 nM [ H]ryanodine. The H1-EVH1 point mutant at 50 nM (H1-W24A) lacked significant RyR1 activity. The data shown are the means  S.E. from at least four replicates. The maximum efficacy of 50 nM GST-H1c was 8-fold, and that of 150 nM H1-EVH1 was 5-fold when tested at 0.5 nM [ H]ryanodine (a concentration of ryanodine below its K ) showing that H1a-EVH1 3 2 is 3-fold less potent than H1c for enhancing [ H]ryanodine binding. d and e show that Homer enhances responsiveness of RyR1 to Ca and caffeine, respectively. SR vesicles (50 g/ml) were incubated as described under “Materials and Methods” for measuring macroscopic Ca fluxes. 2 2 After 160 M Ca was actively loaded into SR vesicles, 8 nM H1c (II) or the same volume of PBS (I) was introduced. After about 5 min, 80 M Ca 2 2 2 2 (d;Ca -induced Ca release) or 10 mM caffeine (e; caffeine-induced Ca release) was added to initiate release of Ca accumulated during the 2 2 loading phase. The initial Ca release rates in the presence (II) or absence (I) of H1c were analyzed and plotted. The initial rate of Ca -induced 2 1 1 2 Ca release increased from 0.002  0.03 to 0.337  0.029 mol mg min in the presence of H1c (n  16). Caffeine-induced Ca release 1 1 increased from 0.009  0.045 to 0.249  0.076 mol mg min in the presence of H1c (n  11). Single Channel Kinetics in Bilayer Lipid Membranes (BLM)—RyR1 was previously shown to accumulate into SR and support active Ca channels reconstitution was performed as described previously (19). loading without forming precipitates (30); however, the presence of The reconstituted channels exhibiting “low P type” gating behavior pyrophosphate buffers extravesicular Ca 10-fold, so that in its pres- 2 th 2 were the focus of the study. RyR1 is extremely sensitive to Cys oxida- ence the increase in free Ca is 1/10 of the total Ca added. tion during preparation of SR (19, 27) and results in 60 –70% of recon- Calcium Imaging of 1B5 Myotubes—The cultured and differentiated stitutions in BLM yielding channels that exhibit “high P type” gating o 1B5 (RyR null) myotubes were transduced with either wild type or behavior, whereas a small fraction of RyR1 channel reconstituted from F1777R/F1782R RyR1 amplicons as previously described (21). Within these preparations exhibit low P type gating behavior (19, 27–29). Low 24 –36 h after transduction, the cells were loaded with the calcium P type channels maintain low open probability over a large range of cis indicator dye Fluo-4 at 37 °C, for 30 min in imaging buffer (125 mM (cytoplasmic) Ca . Because intact muscle possesses a highly reducing NaCl, 5 mM KCl, 2 mM CaCl , 1.2 mM MgSO ,6mM glucose, and 25 mM 2 4 environment, the functional consequences of adding Homer proteins to HEPES, pH 7.4) supplemented with 0.05% bovine serum albumin and the cis chamber focused on reconstituted channels exhibiting low P 5 M Fluo-4 (Molecular Probes Inc., Eugene, OR). The cells were type gating behavior to better reflect the native environment within washed with imaging buffer before being transferred to a Nikon Dia- junctions of intact skeletal muscle (19, 27). Single channel activity was phot microscope. Fluo-4 was excited at 494 nm using the output of a measured at 40 mV (applied cis relative to the trans) using a patch DeltaRam fluorescence imaging system (Photon Technology Interna- clamp amplifier (Dagan 3900). The data were filtered at 1 kHz before tional, Princeton, NJ). Fluorescence emission was measured at 516 nm acquired at 10 kHz by a DigiData 1200A (Axon Inst., Foster City, CA). using a 40 quartz objective. Data presented as the 516 nm emissions The data were analyzed using pClamp 6 (Axon) without additional of Fluo-4 were collected with an intensified CCD camera (Solamere filtering. Average P was calculated from 1 min of recording. 2 2 Technology Group) from individual cells. Caffeine- and K -evoked Ca Macroscopic Ca Flux Measurement—Ca transport across SR transients were generated by exposing the cells for 10 s to increasing membranes was performed using the absorbance dye antipyrylazo III concentrations of caffeine or KCl. Each test was followed by a 30-s wash (30). SR membranes (50 g/ml) were equilibrated at 37 °C in a trans- allowing the intracellular Ca to return to base line. The data were port buffer consisting of 92 mM KCl, 20 mM K-MOPS (pH 7.0), 7.5 mM presented as 1) the percentages of myotubes responding to a given pyrophosphate, and 250 M antipyrylazo III. MgATP (1 mM), 10 g/ml stimulus and 2) the normalized magnitude of the transient, F  F /F creatine phosphokinase, and 5 mM phosphocreatine were present to o o (i.e. F/F ), where F is the mean base-line fluorescence at rest and F is regenerate ATP. Ca flux was monitored by measuring antipyrylazo o o III 710 –790 nm absorbance at 2– 4-s intervals using a diode array the peak fluorescence in response to the stimulus. The data were spectrophotometer (model 8452A, Hewlett Packard). Pyrophosphate recorded from at least 30 myotubes/treatment. Homer Regulates Gain of RyR1 44725 RESULTS AND DISCUSSION Homer Proteins Physically Interact with RyR1—An N-termi- nal EVH1 domain mediates Homer binding to target proteins by contacting specific proline-rich (Homer ligand) sequences found within mGluR1, mGluR5, and the IP R (7). Putative Homer ligand sequences are also present in RyR1 (7). Because Homer proteins are expressed in skeletal muscle (2), we exam- ined the possible in vivo association of Homer with RyR in immunoprecipitation assays from detergent (1% CHAPS) ex- tracts of rat skeletal muscle. Antibodies for Homer1c (H1c), Homer2, and Homer3 co-precipitated RyR1, whereas the pre- immune sera failed to do so (Fig. 1a). The interaction of RyR1 with Homer proteins was independently confirmed using GST pull-down assays with GST-Homer1c and GST-Homer3 fusion protein and detergent extracts of skeletal muscle (RyR1) (Fig. 1b; lanes GST-H1c and GST-H3). To examine the presence of endogenous Homer proteins in commonly used preparations of junctional SR enriched in RyR1, we isolated several membrane preparations from rabbit skeletal muscle by sucrose density gradient centrifugation and probed them by Western blot anal- ysis with RyR1- and Homer-specific antibodies (Fig. 1c). In every junctional SR preparation tested, H1c co-purified with RyR1, although the relative amounts of the H1c varied from preparation to preparation. Homer3 was also co-purified with RyR1 but was not detected in every junctional SR preparation examined (Fig. 1c). In all four preparations that were analyzed, H1c and Homer3 proteins were less enriched than RyR1. Be- cause Homer is a cytosolic protein, these results are consistent with the physical association of Homer with RyR1 and indicate that the co-enrichment of Homer relative to RyR1 varies from preparation to preparation. Presumably, Homer proteins vari- ably dissociated from the RyR1 complex during purification of junctional SR membranes. H1C and H1-EVH1 Enhance RyR1 Gating—To examine the functional consequence of the protein-protein interaction, we determined whether Homer could modify RyR1 channel gating behavior using the BLM method. Single RyR1 channels were reconstituted with an artificial BLM by inducing fusion of purified junctional SR vesicles. Those channels exhibiting the low P gating mode (19, 27–29) were selected for examining the influence of exogenous Homer protein added to the cytoplasmic (cis) side of the channel measured under voltage clamp condi- tions. Both the long form H1c (Fig. 2a) and short form H1- EVH1 lacking the coiled-coil domain (Fig. 2b) were highly potent and efficacious toward enhancing RyR1 channel open probability (P ). With 5 nM H1c added to the cis chamber, P o o increased 10-fold. H1-EVH1 was equally effective but 7-fold less potent (34 nM H1-EVH1 needed to enhance P 10-fold). The murine homolog of Ena (Mena) possesses an EVH1 domain that is structurally similar to Homer but binds a distinct pro- line-rich sequence (7). Mena lacked detectable activity on chan- nel P at the highest concentration tested (50 nM). Both H1- EVH1 and H1c shortened channel closed dwell time by over 100-fold without significantly altering open dwell time (data not shown). We also examined the effects of the immediate- early gene product Homer1a-EVH1 on high P gating mode channels reconstituted in BLM. Homer1a-EVH1 (40 nM) signif- chard analysis reveals that H1c increases maximum receptor occupancy (B ) 3-fold without altering affinity (K )(a and b; data points are the max D means  S.D. from two independent experiments each performed in FIG.3. Homer increases the maximum density of high affinity triplicate). c, H1c enhances the efficacy of Ca toward activating high 3 3 [ H]ryanodine binding and amplifies to junctional SR. a, equilib- affinity [ H]Ry binding 2.5-fold compared with control, without altering rium binding curves of [ H]ryanodine and 50 g/ml junctional SR were the half-activation constant (EC  210 versus 280 nM, respectively; performed using competition with unlabeled ryanodine (cold titration). mean of n  8). Free Ca concentration was adjusted by the addition The presence of 50 nM GST-H1c (H1C) increased occupancy over the of CaCl and EGTA based on calculations from Bound and Determined entire dose-response curve compared with the GST control. b, Scat- software (53). 44726 Homer Regulates Gain of RyR1 biochemical indicator of modulation of the open probability of the channel by exogenously added ligands (24 –26). When GST was included in the binding medium, occupancy of [ H]ryano- dine was indistinguishable from control (Fig. 2c). Similarly, [ H]ryanodine binding was unaltered by inclusion of Mena (100 nM). In marked contrast, 50 nM H1c or its fusion protein, GST-H1c, enhanced the binding of 5 nM [ H]ryanodine by nearly 5-fold (Fig. 2c). The actions of GST-H1c measured in the presence [ H]ryanodine at a concentration well below its K (0.5 nM) resulted in 8-fold enhancement of occupancy (Fig. 2c, right panel) consistent with the effects of Homer protein on enhancing channel P in BLM studies (Fig. 2a). At a concen- tration of 5 nM [ H]ryanodine, the addition of 50 nM H1-EVH1 mimicked the activity of full-length H1c but exhibited smaller (2-fold) but statistically significant (p  0.05) enhancement in binding compared with control. When tested at subsaturating [ H]ryanodine (0.5 nM), the addition of 150 nM H1-EVH1 in- creased binding 5-fold, whereas 50 nM H1c increased binding 8-fold (Fig. 2c). This demonstrates that both forms have a similar ability to enhance RyR1 activity consistent with their ability to enhance the P of channels reconstituted in BLM (Fig. 2b). The 3-fold difference in the potency observed with the long and short forms of Homer on RyR1 could be attributed to the multivalency of H1c that may optimize conformational interaction with RyR1 oligomeric units. As a further control, we found that the W24A point mutant of H1-EVH1, which does not bind Homer ligands (7), had negligible activity on altering the binding of [ H]ryanodine to RyR1 under the stated assay condi- tion (Fig. 2c). These results support the hypothesis that specific protein-protein interactions between Homer proteins and RyR1 can variably enhance the gain of signaling events mediated 2 2 2 through Ca -induced Ca release (CICR), and hence Ca released from SR, by influencing the fraction of RyR channels within junctional SR responsive to physiological activators. To further test the hypothesis, [ H]ryanodine-binding curves were performed in the presence of nearly optimal Ca for channel activation (1 M) and in the absence or presence of Homer protein (Fig. 3a). Scatchard analysis revealed that H1c enhanced the binding of [ H]ryanodine over the complete range of concentrations, enhancing maximum occupancy 3-fold (from B  4.2 and 12.7 pmol mg in the absence and presence of max 50 nM H1c, respectively) without significantly altering binding affinity (K  7.95 and 9.06 nM, respectively; Fig. 3b). These results indicate that formation of H1c-RyR1 complexes in vitro significantly increased the density of Ca -responsive channels within the SR membrane. Because Homer protein appears to dissociate from SR preparations during biochemical enrich- FIG.4. Enhancement of RyR1 channel function by Homer is ment of RyR1, the ability of exogenous Homer to increase the specifically blocked by competing peptide. H1c was preincubated density of functional RyR1 able to bind ryanodine in a Ca - with 1000 synthetic wild type Homer-binding peptide (Pept ), syn- wt dependent manner likely results from formation of new pro- thetic peptide missing the critical phenylalanine (Pept ), or PBS at mut 37 °C for 10 min. After completion of preloading of SR with 160 M tein-protein complexes that may have been lost during prepa- Ca , Homer in the presence or absence of Pept or Pept was wt mut ration of junctional SR membranes. introduced. The final concentration of the H1c in the transport buffer Although in skeletal muscle CICR is not necessary to initiate was 20 nM (a)or8nM (b and c). 10 M ryanodine (Ry) or Ruthenium Red E-C coupling, nor is it necessary to engage retrograde signaling (RR) was added to activate or inhibit RyR1, whereas 0.5 M thapsigar- gin (TG) was introduced to inhibit SR Ca pumps. In b and c, the final (32), it is generally agreed that Ca is an important physio- 2 2 concentration of Ca used to induce Ca release was 60 M. Summary logical modulator of RyR1 in skeletal muscle. Several ligands data represent the mean of 21 (a),6(b), or 6 (c) repetitions of these have been shown to influence the sensitivity of RyR to activa- experiments. 2 3 tion by Ca (24, 26). Using [ H]ryanodine binding analysis near K for the radioligand, H1c (50 nM) did not significantly icantly enhanced the gating activity of in the presence of 7 M shift the sensitivity of RyR1 to activation by Ca relative to cis Ca (4-fold; not shown). control (EC  210 versus 280 nM Ca , respectively; Fig. 3c). Mechanism of Homer Modulation of RyR1—The manner in which Homer proteins enhances RyR1 activity was examined However, H1c did enhance maximal occupancy 2.5-fold, indi- cating an enhanced “gain” of RyR1 toward activation by Ca in more detail by measuring their influence on the binding of 3 3 [ H]ryanodine to skeletal muscle JSR. Because [ H]ryanodine consistent with an increase in the density of Ca -responsive RyR1 complexes. binds with high affinity to an open state of the RyR channel, occupancy of [ H]ryanodine-binding sites provides a convenient Homer Enhances CICR and Caffeine—A functional corre- Homer Regulates Gain of RyR1 44727 FIG.5. Expression of RyR1 possessing a mutation of a putative Homer-ligand sequence in 1B5 myotubes. a shows that 1B5 myotubes constitutively express immunoreactive Homer protein. The upper panel shows 1B5 cells expressing wt RyR1 immunostained with pan Homer antibody as described under “Materials and Methods” at 10 magnification. The same results were observed with 1B5 myotubes expressing RyR1 possessing point mutation F1777R/F1782R. The lower panel shows results from Western analysis of the whole membrane fraction obtained from 1B5 myotubes and rabbit skeletal muscle junctional SR probed with Homer1c-selective antibody. b, immunocytochemical analysis of 1B5 myotubes at 10 before (left panel) and after transduction with HSV-1 virions containing wild type (wt) RyR1 (middle panel) and full-length RyR1 containing a point mutation within the putative Homer ligand domain (HLM, right panel). 100 magnification inserts in the middle and right panels show that both wild type and HLM RyR1 have the characteristic punctate staining pattern of RyR1 when it is targeted properly to peripheral junctions. late to the results obtained from radioligand receptor binding siveness of RyR1 at the level of the single channel and under- analysis was undertaken to understand how H1c influences lies the increased “gain” of RyR1-mediated Ca efflux units 2 2 macroscopic Ca efflux elicited by either Ca (CICR) or within junctional SR (defined by two independent measures, 2 3 caffeine from actively loaded skeletal muscle SR vesicles (Fig. macroscopic Ca efflux (CICR) and [ H]ryanodine binding 4, b and c). This assay utilizes pyrophosphate to support density). These actions of Homer protein exhibited a high de- active loading of Ca into the vesicles (30). After calcium gree of molecular specificity and were mediated by their unique loading, the addition of H1c to a subthreshold concentration EVH1 domain. (8 nM) that did not directly initiate net Ca efflux from the RyR1 Homer-binding Domain Regulates Gain—Conforma- SR vesicles enhanced the initial rate of RyR1-mediated tional changes in IP R caused by IP binding and store deple- 3 3 Ca elicited by subsequent addition of either optimal 80 M tion was shown to provide a retrograde signal to opening of 2 2 2 total Ca (8.0 M free Ca ) or saturating caffeine (10 mM) store-operated Ca channels in the plasma membrane (33, 7.9- and 8.2-fold, respectively. Thus, H1c enhanced the max- 34), similar to the bi-directional signaling between RyR1 and imum efficacy of both Ca and caffeine for activation of  -DHPR in skeletal muscle that is essential for engaging E-C 1S RyR1. coupling (17). Moreover, evidence for conformational coupling Homer Modulation of RyR1 Is Blocked by Specific Peptide— between RyR and store-operated Ca channels has been re- The specificity of H1c for enhancing Ca release from SR was cently presented (35, 36). Thus, conformational coupling ap- examined by preincubating H1c with a synthetic “blocking” pears to be a fundamental mechanism involved in generating peptide that mimics the Homer ligand within the C terminus of localized Ca signals. Both RyR and  -DHPR possess dis- 1S mGluR-1 (1). Fig. 4a (trace I) demonstrates that addition of crete polyproline sequences that are putative Homer ligands. saturating (20 nM) H1c induced net Ca release from actively RyR1 contains a putative Homer-ligand domain between amino loaded SR vesicles. In the absence of added Homer, Ca was acids 1773 and 1783. A second putative Homer binding domain not released until the addition of either ryanodine (10 M; trace can be found in RyR1 (PPPGYA; amino acids 804 – 809), where III) or the SR Ca pump inhibitor thapsigargin (0.5 M; trace the essential hydrophobic phenylalanine is substituted with IV). The effect of H1c could be blocked by the addition of 50 M tyrosine. wild type peptide (Pept ) but not by a point mutant of the We therefore tested the hypothesis that Homer is involved in wt blocking peptide (Pept ) that lacks Homer binding activity modulating the strength of depolarized-induced Ca release mut (1) (Fig. 4a, trace II). Pept does not block release by subse- in intact cells. 1B5 dyspedic skeletal myotubes express all the wt quent addition of ryanodine. Pept , but not Pept , also pre- proteins known to be associated with the mature triadic junc- wt mut vented the ability of H1c to amplify CICR after active loading of tion but lack expression of the three RyR isoforms (23, 37). 160 M Ca into SR (Fig. 4, b and c). CICR induced by the Transduction of 1B5 myotubes with wild type RyR1 cDNA addition of 60 M Ca in the presence of 8 nM H1c exhibited reconstituted bi-directional signaling between RyR1 and  - 1S indistinguishable rates of Ca release in the presence or ab- DHPR and directed the organization of  -DHPR particles 1S sence of Pept (Fig. 4c, traces I and II). Subsequent addition into organized arrays of tetrads (38, 39). Homer1 protein was mut of Ruthenium Red (10 M), an RyR channel blocker, resulted in detected in 1B5 myotubes by immunocytochemical and West- active re-uptake of Ca into SR vesicles. However, in the ern blot analyses (Fig. 5a). presence of Pept (Fig. 4b, trace III), the addition of 60 M The possible functional role of one of the putative Homer- wt 2 2 2 Ca displayed a net Ca uptake into SR because SR Ca binding sites of RyR1 was tested by incorporating site-directed pump-transport into the vesicles surpassed the amount of re- mutations (F1777R/F1782R) within the full-length protomer. lease through RyR1. Active accumulation could be interrupted The substitutions were chosen based on the recent data show- abruptly and was quickly followed by Ca release after sub- ing that hydrophobic phenylalanine C-terminal to polyprolines sequent addition of 10 M ryanodine (Fig. 4, b and c, trace IV). are essential for Homer binding (7). RyR protomers from 1B5 As further evidence of its specificity, we confirmed that Pept myotubes expressing full-length wild type and F1777R/F1782R wt did not affect modulation of RyR1 by the scorpion toxin mau- RyR1 proteins exhibited the same mobility by SDS-PAGE (not rocalcine, a potent activator of RyR1 (31) (not shown). shown) and show similar levels of expression by immunocyto- Taken together, these results revealed that physical associ- chemistry. At higher magnification both wt and F1777R/ ation of H1c with RyR1 dramatically enhanced Ca respon- F1782R RyR1 exhibited the characteristic punctate pattern of 44728 Homer Regulates Gain of RyR1 FIG.6. Mutation of a putative Homer-ligand sequence of RyR1 attenuates excitation-contraction coupling in 1B5 myotubes. a shows the responses of 1B5 myotubes expressing wild type and the putative HLM (F1777R/F1782R RyR1) to threshold (1 mM) and saturating (30 mM) caffeine. 40% fewer myotubes responded to the threshold concentration of caffeine, whereas all myotubes responded to saturating caffeine. The normalized amplitude of responses to threshold caffeine were 56% smaller (p  0.001) in HLM versus wild type but were not different at saturating caffeine. The data were analyzed from 68 and 92 wild type and HLM myotubes, respectively. c and d show the K -evoked Ca transients following Homer Regulates Gain of RyR1 44729 expression within the myotubes, which has previously been The present findings reveal that Homer proteins through shown to be consistent with targeting of RyR protein to periph- their direct interaction with RyR1 can modulate the gain of E-C eral junctions (Fig. 5b) (38, 39). coupling. This can occur in three ways. First, the interaction of Both wild type and the putative Homer ligand mutation constitutively expressed H1c could bind to one to four channel (HLM; F1777R/F1782R) RyR1 responded to caffeine chal- protomers to regulate cooperativity associated with channel lenges. However, the percentage of HLM myotubes responding activation. Second, H1c multimers could cross-link multiple to a threshold concentration of caffeine (1 mM) was 39.6% lower RyR1 oligomers to facilitate coupled gating among channels than wild type myotubes (43.5% versus 72.0% of n  92 and 68 elicited by FKBP12 (40). Because localized Ca sparks occur myotubes, respectively; Fig. 6a, left bars). When challenged upon release of Ca through a cluster of RyR (41), it is inter- with saturating caffeine (30 mM), the frequency of responses esting to note that H1c has been recently shown to induce with HLM and wild type myotubes were both 100%. The nor- sparks in frog skeletal muscle (42). Homer-RyR associations malized magnitude of the Fluo-4 fluorescence signal (F/F )isa o may therefore be a significant physiological regulator of spark quantitative measure of the amount of Ca mobilized in re- activity that contributes to Ca waves that sweep through sponse to direct activation of release channels by caffeine. The cells and subsequently activate myocyte contraction. Third, magnitude of the responses to 1 mM caffeine was 56.6% smaller multimeric H1c could directly cross-link RyR1 with  -DHPR 1S (F/F  6.8% versus 15.7%; p  0.003) in myotubes expressing o to enhance the efficiency of E-C coupling in a manner already HLM. However, 30 mM caffeine elicited Ca responses whose described for mGluR1 and IP R (1, 7). Whether Homer inter- magnitude were indistinguishable between wild type and HLM actions with a putative binding site on  -DHPR contribute to 1S myotubes, indicating that the SR stores of both cell types were modulation of E-C coupling remains to be determined. filled with Ca to similar levels (Fig. 6a, right bars). Homer short form (i.e. H1a-EVH1) is encoded by a prema- Expression of either wild type or HLM cDNAs in 1B5 myo- turely terminated transcript that lacks the coiled-coil domain tubes also restored Ca responses to K depolarization con- that is required for the formation of Homer multimers. The sistent with restoration of skeletal type excitation-contraction currently favored mechanism for how Homer proteins regulate coupling, and the amplitude of this response increased in a signaling functions suggests that the H1-EVH1, an immediate- saturable manner with increasing K concentration between early gene product, is rapidly expressed in response to cellular 10 and 100 mM (Fig. 6, c and d). The expressed proteins pro- activity, whereas long form H1c is constitutively expressed. In duced Ca transients that were kinetically identical, and the this scheme, H1c plays an adapter function, linking target apparent potency for producing K -evoked Ca transients proteins such as mGluR1 and IP R in neurons. Immediate- was unaltered by the mutation (EC  26.6  1.2 and 27.0 early expression of H1-EVH1 acts in a dominate-negative fash- 1.2 mM for wild type and mutant protein, respectively). Impor- ion by competing with existing H1c complexes, thereby impair- tantly, however, the maximum amplitude of the K -evoked ing the formation and maintenance of Homer-mediated cluster Ca transients produced by cells expressing HLM were 33% of protein complexes (2). However, experimental evidence for a smaller than those produced by wild type RyR1 (mean F/F more dynamic role for Homer proteins in cellular regulation 76  0.85 and 58  2.0; n  34 and 48 cells for wild type and has recently been defined (13, 43– 45). For example, experi- HLM, respectively; p  0.001), demonstrating Homer’s impor- ments using single particle tracking demonstrated that the tance in modulating depolarization induced Ca release. The composition of Homer-mediated clustering of protein com- possible existence of a second Homer ligand domain within plexes is highly dynamic. The exchange of the proteins between RyR1 (amino acids 804 – 809) and its functional role remains to dispersed and clustered states is fast and is regulated during be investigated. physiological processes (44, 45). The present results suggest Functional Significance of Interactions between RyR1 and that the catalytic activity of Homer proteins toward RyR may Homer Proteins—The present study demonstrates that Homer provide one mechanism linking Ca signals with dynamic binding directly enhances responses of RyR1 to physiological regulation of Homer-mediated protein clustering. and pharmacological stimuli. This activity is conferred by the One anticipated consequence of Homer catalytic activity is to EVH1 domain and does not require the cross-linking adapter increase the spatial fidelity of signaling beyond what can be function of Homer. However, multimeric H1c does appear to achieved by simple physical coupling. Homer and RyR proteins significantly enhance the potency toward enhancing RyR1 re- are widely expressed, and it may be anticipated that their sponses. Homer is the first example of an “adapter” that also physical and functional interactions identified here contribute modifies signaling properties of its target protein. Multi-PDZ to temporal and spatial patterning of intracellular Ca signal- domain proteins are reported to activate ion channels, but this ing. In skeletal muscle, release of Ca through RyR1 is the key activity is mediated by simple physical cross-linking (14). The event linking membrane depolarization and mechanical activ- nanomolar affinities required for channel modulation with full- ity during E-C coupling. In neurons, Homer proteins play a length RyR and the complete long and short forms of Homer special role in adaptation to cellular activity. CICR has been indicate a structurally specific interaction. Previous studies demonstrated in a number of neuronal cell types (46 – 48). that used peptides to block the binding of EVH domains with Evidence for the role of RyR in the regulation of specific aspects their target proteins suggested affinities in the micromolar of neuronal plasticity, dendritic growth, and spatial learning range (1, 8). Because our studies used full-length proteins, it is has recently been demonstrated (49 –52). The present results inferred that additional structure is important for optimizing predict that RyR1-Homer interactions may also contribute el- these binding interactions. 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