To investigate the mechanism of regulation of sarco-endoplasmic reticulum Ca 2؉ -ATPase (SERCA) by phospholamban (PLB), we expressed Cerulean-SERCA and yellow fluorescent protein (YFP)-PLB in adult rabbit ventricular myocytes using adenovirus vectors. SERCA and PLB were localized in the sarcoplasmic reticulum and were mobile over multiple sarcomeres on a timescale of tens of seconds. We also observed robust fluorescence resonance energy transfer (FRET) from Cerulean-SERCA to YFP-PLB. is a P-type ion pump that maintains the Ca 2ϩ gradient across the endoplasmic reticulum. In cardiac muscle cells, calcium (Ca 2ϩ ) sequestration by SERCA is critical for muscle relaxation during the cardiac cycle, and disordered Ca 2ϩ handling is associated with cardiac dysfunction (1-3). SERCA activity is regulated by phospholamban (PLB), a helical transmembrane peptide that reduces the apparent affinity of the pump for Ca 2ϩ . Inhibition of SERCA by PLB is partially relieved through phosphorylation of PLB by protein kinase A and Ca 2ϩ /calmodulindependent protein kinase (4), which alters the structure of the PLB-SERCA regulatory complex (5) and increases PLB oligomerization into non-inhibitory pentamers (5, 6). It is widely recognized that PLB inhibition of SERCA is also relieved by elevated Ca 2ϩ , but the mechanism of this functional effect is unclear. One possibility is that PLB binds selectively to the Ca 2ϩ -free "E2" conformation of SERCA and cannot bind to the Ca 2ϩ -bound "E1" conformation (Fig. 1A, Dissociation Model). This model is supported by the observation that elevated Ca 2ϩ abolishes chemical cross-linking of the PLB transmembrane domain to reactive residues on SERCA (7-11) and reduces coimmunoprecipitation (12). Cross-linking was also prevented by the SERCA inhibitor thapsigargin (Tg) (7, 9 -11). Another recent study showed that oligomerization of a PLB-SERCA fusion construct was increased by micromolar Ca 2ϩ (13), consistent with the idea that Ca 2ϩ causes displacement of PLB from the inhibitory cleft, permitting PLB self-association into pentamers. Overall, these data suggest that only certain conformational substates of SERCA interact with PLB. The dissociation model predicts that PLB unbinds from SERCA during the period of systole (cardiac contraction) when Ca 2ϩ is high, and the regulatory complex reforms during diastole (cardiac relaxation) when the cytoplasmic Ca 2ϩ concentration is low. An alternative theory was generated from in vitro measurements of fluorescence resonance energy transfer (FRET) between SERCA and PLB. Mueller et al. (14) showed that FRET was decreased, but not abolished, by Ca 2ϩ . This result suggested that relief of inhibition was accomplished by a conformational change of the regulatory complex, rather than unbinding of PLB from the pump. This study estimated a dissociation constant significantly lower than the expected in vivo concentrations of PLB and SERCA. Li et al. (15) also provided evidence from fluorescence spectroscopy that suggests that the binding of PLB and Ca 2ϩ is not...

We have used a “2-color” SERCA (sarco/endoplasmic reticulum calcium ATPase) biosensor and a unique high-throughput fluorescence lifetime plate-reader (FLT-PR) to develop a high-precision live-cell assay designed to screen for small molecules that perturb SERCA structure. A SERCA construct, in which red fluorescent protein (RFP) was fused to the N terminus and green fluorescent protein (GFP) to an interior loop, was stably expressed in an HEK cell line that grows in monolayer or suspension. Fluorescence resonance energy transfer (FRET) from GFP to RFP was measured in the FLT-PR, which increases precision 30-fold over intensity-based plate-readers without sacrificing throughput. FRET was highly sensitive to known SERCA modulators. We screened a small chemical library and identified ten compounds that significantly affected 2-color SERCA FLT. Three of these compounds reproducibly lowered FRET and inhibited SERCA in a dose-dependent manner. This assay is ready for large-scale HTS campaigns, and is adaptable to many other targets.

The E3 ubiquitin ligase atrophin interacting protein 4 (AIP4) mediates ubiquitination and down-regulation of the chemokine receptor CXCR4. AIP4 belongs to the Nedd4-like homologous to E6-AP carboxy terminus domain family of E3 ubiquitin ligases, which typically bind proline-rich motifs within target proteins via the WW domains. The intracellular domains of CXCR4 lack canonical WW domain binding motifs; thus, whether AIP4 is targeted to CXCR4 directly or indirectly via an adaptor protein remains unknown. Here, we show that AIP4 can interact directly with CXCR4 via a novel noncanonical WW domain-mediated interaction involving serine residues 324 and 325 within the carboxy-terminal tail of CXCR4. These serine residues are critical for mediating agonist-promoted binding of AIP4 and subsequent ubiquitination and degradation of CXCR4. These residues are phosphorylated upon agonist activation and phosphomimetic mutants show enhanced binding to AIP4, suggesting a mechanism whereby phosphorylation mediates the interaction between CXCR4 and AIP4. Our data reveal a novel noncanonical WW domain-mediated interaction involving phosphorylated serine residues in the absence of any proline residues and suggest a novel mechanism whereby an E3 ubiquitin ligase is targeted directly to an activated G protein-coupled receptor.

Phospholamban (PLB) oligomerization, quaternary structure, and sarco(endo)plasmic reticulum calcium ATPase (SERCA) binding were quantified by fluorescence resonance energy transfer (FRET) in an intact cellular environment. FRET between cyan fluorescent protein-PLB and yellow fluorescent protein-PLB in AAV-293 cells showed hyperbolic dependence on protein concentration, with a maximum efficiency of 45.1 ؎ 1.3%. The observed FRET corresponds to a probe separation distance of 58.7 ؎ 0.5 Å , according to a computational model of intrapentameric FRET. This is consistent with models of the PLB pentamer in which cytoplasmic domains fan out from the central bundle of transmembrane helices. An I40A mutation of PLB did not alter pentamer conformation but increased the concentration of half-maximal FRET (K D ) by >4-fold. This is consistent with the previous observation that this putatively monomeric mutant still oligomerizes in intact membranes but forms more dynamic pentamers than wild type PLB. PLB association with SERCA, measured by FRET between cyan fluorescent protein-SERCA and yellow fluorescent protein-PLB, was increased by the I40A mutation without any detectable change in probe separation distance. The data indicate that the regulatory complex conformation is not altered by the I40A mutation. A naturally occurring human mutation (L39Stop) greatly reduced PLB oligomerization and SERCA binding and caused mislocalization of PLB to the cytoplasm and nucleus. Overall, the data suggest that the PLB pentamer adopts a "pinwheel" shape in cell membranes, as opposed to a more compact "bellflower" conformation. I40A mutation decreases oligomerization and increases PLB binding to SERCA. Truncation of the transmembrane domain by L39Stop mutation prevents anchoring of the protein in the membrane, greatly reducing PLB binding to itself or its regulatory target, SERCA.The 52-amino acid protein phospholamban (PLB) 2 is an important regulator of cardiac calcium handling (1). PLB binds avidly (2) but reversibly (3) to the sarco(endo)plasmic reticulum calcium ATPase (SERCA), reducing this calcium pump's affinity for calcium (4). PLB also oligomerizes into pentamers through leucine zipper interactions in its transmembrane domain (5, 6). NMR studies indicate that PLB tertiary structure consists of an N-terminal (cytosolic) ␣-helix (domain IA) connected by a flexible loop (domain IB) to a second ␣-helix (domain II) that is anchored in the membrane of the sarcoplasmic reticulum (7). Several possible configurations of the cytoplasmic domains of pentameric PLB have been described. Some of these portray the domain IA ␣-helix as being nearly normal to the surface of the membrane (8, 9), whereas others indicate axial declination of domain IA (7, 10 -14), permitting contact with the surface of the membrane (15, 16). These previous studies were performed using in vitro preparations of PLB in defined lipids or detergent. To investigate the quaternary structure of PLB in the membranes of living cells and distinguish between these structural mo...

Calcium (Ca2+) dysregulation is a hallmark of heart failure and is characterized by impaired Ca2+ sequestration into the sarcoplasmic reticulum (SR) by the SR-Ca2+-ATPase (SERCA). We recently discovered a micropeptide named DWORF (DWarf Open Reading Frame) that enhances SERCA activity by displacing phospholamban (PLN), a potent SERCA inhibitor. Here we show that DWORF has a higher apparent binding affinity for SERCA than PLN and that DWORF overexpression mitigates the contractile dysfunction associated with PLN overexpression, substantiating its role as a potent activator of SERCA. Additionally, using a well-characterized mouse model of dilated cardiomyopathy (DCM) due to genetic deletion of the muscle-specific LIM domain protein (MLP), we show that DWORF overexpression restores cardiac function and prevents the pathological remodeling and Ca2+ dysregulation classically exhibited by MLP knockout mice. Our results establish DWORF as a potent activator of SERCA within the heart and as an attractive candidate for a heart failure therapeutic.