Rod Visual Pigment Optimizes Active State to Achieve Efficient G Protein Activation as Compared with Cone Visual Pigments

Most vertebrate retinas contain two types of photoreceptor cells, rods and cones, which show different photoresponses to mediate scotopic and photopic vision, respectively. These cells contain different types of visual pigments, rhodopsin and cone visual pigments, respectively, but little is known about the molecular properties of cone visual pigments under physiological conditions, making it difficult to link the molecular properties of rhodopsin and cone visual pigments with the differences in photoresponse between rods and cones. Here we prepared bovine and mouse rhodopsin (bvRh and mRh) and chicken and mouse green-sensitive cone visual pigments (cG and mG) embedded in nanodiscs and applied time-resolved fluorescence spectroscopy to compare their G_t activation efficiencies. Rhodopsin exhibited greater G_t activation efficiencies than cone visual pigments. Especially, the G_t activation efficiency of mRh was about 2.5-fold greater than that of mG at 37 °C, which is consistent with our previous electrophysiological data of knock-in mice. Although the active state (Meta-II) was in equilibrium with inactive states (Meta-I and Meta-III), quantitative determination of Meta-II in the equilibrium showed that the G_t activation efficiency per Meta-II of bvRh was also greater than those of cG and mG. These results indicated that efficient G_t activation by rhodopsin, resulting from an optimized active state of rhodopsin, is one of the causes of the high amplification efficiency of rods.     

The processed isoform of the translation termination factor eRF3 localizes to the nucleus to interact with the ARF tumor suppressor

The eukaryotic releasing factor eRF3 is a multifunctional protein that plays pivotal roles in translation termination as well as the initiation of mRNA decay. eRF3 also functions in the regulation of apoptosis; eRF3 is cleaved at Ala73 by an as yet unidentified protease into processed isoform of eRF3 (p-eRF3), which interacts with the inhibitors of apoptosis proteins (IAPs). The binding of p-eRF3 with IAPs leads to the release of active caspases from IAPs, which promotes apoptosis. Although full-length eRF3 is localized exclusively in the cytoplasm, p-eRF3 localizes in the nucleus as well as the cytoplasm. We here focused on the role of p-eRF3 in the nucleus. We identified leptomycin-sensitive nuclear export signal (NES) at amino acid residues 61–71 immediately upstream of the cleavage site Ala73. Thus, the proteolytic cleavage of eRF3 into p-eRF3 leads to release an amino-terminal fragment containing NES to allow the relocalization of eRF3 into the nucleus. Consistent with this, p-eRF3 more strongly interacted with the nuclear ARF tumor suppressor than full-length eRF3. These results suggest that while p-eRF3 interacts with IAPs to promote apoptosis in the cytoplasm, p-eRF3 also has some roles in regulating cell death in the nucleus.     

Expression profiles of genes involved in apoptosis and selenium metabolism in articular cartilage of patients with Kashin–Beck osteoarthritis

Kashin–Beck disease (KBD) is a special type of endemic osteoarthritis. It has been suggested that alterations in selenium metabolism and apoptosis play a role in KBD. However, the underlying molecular mechanism remains largely unclear. We performed a microarray analysis using RNA isolated from cartilages of KBD patients and healthy controls, through Significance Analysis of Microarray (SAM) software. Functional gene networks and crucial molecules associated with differentially expressed genes were investigated via Ingenuity Pathway Analysis (IPA) and hub gene analysis. Quantitative real-time PCR was used to check the validation of chip test. We identified 52 up-regulated apoptosis-related genes and 26 down-regulated selenium-related genes between KBD and controls, and these genes associated with the “MYC-mediated apoptosis signaling pathway”. We confirmed the results from array studies with quantitative real-time PCR analysis. Our results suggest that abnormal regulation of selenium metabolism and apoptosis through the MYC mediated signaling pathway contributes to the pathogenesis of KBD, but the relationship between apoptosis gene and selenium gene was not found.     

Extracellular Calcium Modulates Actions of Orthosteric and Allosteric Ligands on Metabotropic Glutamate Receptor 1α

Metabotropic glutamate receptor 1α (mGluR1α), a member of the family C G protein-coupled receptors, is emerging as a potential drug target for various disorders, including chronic neuronal degenerative diseases. In addition to being activated by glutamate, mGluR1α is also modulated by extracellular Ca²⁺. However, the underlying mechanism is unknown. Moreover, it has long been challenging to develop receptor-specific agonists due to homologies within the mGluR family, and the Ca²⁺-binding site(s) on mGluR1α may provide an opportunity for receptor-selective targeting by therapeutics. In the present study, we show that our previously predicted Ca²⁺-binding site in the hinge region of mGluR1α is adjacent to the site where orthosteric agonists and antagonists bind on the extracellular domain of the receptor. Moreover, we found that extracellular Ca²⁺ enhanced mGluR1α-mediated intracellular Ca²⁺ responses evoked by the orthosteric agonist l-quisqualate. Conversely, extracellular Ca²⁺ diminished the inhibitory effect of the mGluR1α orthosteric antagonist (S)-α-methyl-4-carboxyphenylglycine. In addition, selective positive (Ro 67-4853) and negative (7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester) allosteric modulators of mGluR1α potentiated and inhibited responses to extracellular Ca²⁺, respectively, in a manner similar to their effects on the response of mGluR1α to glutamate. Mutations at residues predicted to be involved in Ca²⁺ binding, including E325I, had significant effects on the modulation of responses to the orthosteric agonist l-quisqualate and the allosteric modulator Ro 67-4853 by extracellular Ca²⁺. These studies reveal that binding of extracellular Ca²⁺ to the predicted Ca²⁺-binding site in the extracellular domain of mGluR1α modulates not only glutamate-evoked signaling but also the actions of both orthosteric ligands and allosteric modulators on mGluR1α.     

Polymorphisms in the Human Inhibitory Signal-regulatory Protein α Do Not Affect Binding to Its Ligand CD47

CD47 is a widely distributed membrane protein that interacts with signal-regulatory protein α (SIRPα), an inhibitory receptor on myeloid cells that gives a “don''t-eat-me” signal. Manipulation of the interaction is of considerable interest in the immunotherapy of cancer and in xenotransplantation. The amino-terminal ligand binding domain of SIRPα is highly polymorphic in contrast to the single Ig-like domain of CD47. There is confusion as to whether the polymorphisms will affect ligand binding, but this is an important point for this interaction and other paired receptors being considered as targets for therapy. We show by x-ray crystallography that one human SIRPα allele differing in 13 amino acid residues has a very similar binding site and that several different alleles all bind CD47 with similar affinity as expected because the residues are mostly surface-exposed and distant from the binding site. A peptide from the binding site of CD47 has been reported to mimic the CD47 interaction with SIRPα, but we could find no binding. We discuss the possible pitfalls in determining the affinity of weak interactions and also speculate on how SIRPα polymorphisms may have been selected by pathogens and how this may also be true in other paired receptors such as the KIRs.     

Characterization of the Deubiquitinating Activity of USP19 and Its Role in Endoplasmic Reticulum-associated Degradation

Deubiquitinating enzymes (DUBs) regulate various cellular processes ranging from protein degradation to cellular signaling. USP19, the only DUB containing a carboxyl-terminal transmembrane domain, was proposed to function in endoplasmic reticulum-associated degradation (ERAD). Here we characterize the function and regulation of USP19. We identify Hsp90 as a specific partner that binds the catalytic domain of USP19 to promote substrate association. Intriguingly, although overexpressed USP19 interacts with Derlin-1 and other ERAD machinery factors in the membrane, endogenous USP19 is mostly in the cytosol where it binds Hsp90. Accordingly, we detect neither interaction of endogenous USP19 with Derlin-1 nor significant effect on ERAD by USP19 depletion. The USP19 transmembrane domain appears to be partially stabilized in the cytosol by an interaction with its own catalytic domain, resulting in auto-inhibition of its deubiquitinating activity. These results clarify the role of USP19 in ERAD and suggest a novel DUB regulation that involves chaperone association and membrane integration. Moreover, our study indicates that the localization of tail-anchored membrane proteins can be subject to regulation in cells.     

Generating Symmetry in the Asymmetric ATP-binding Cassette (ABC) Transporter Pdr5 from Saccharomyces cerevisiae

Pdr5 is a plasma membrane-bound ABC transporter from Saccharomyces cerevisiae and is involved in the phenomenon of resistance against xenobiotics, which are clinically relevant in bacteria, fungi, and humans. Many fungal ABC transporters such as Pdr5 display an inherent asymmetry in their nucleotide-binding sites (NBS) unlike most of their human counterparts. This degeneracy of the NBSs is very intriguing and needs explanation in terms of structural and functional relevance. In this study, we mutated nonconsensus amino acid residues in the NBSs to its consensus counterpart and studied its effect on the function of the protein and effect on yeast cells. The completely “regenerated” Pdr5 protein was severely impaired in its function of ATP hydrolysis and of rhodamine 6G transport. Moreover, we observe alternative compensatory mechanisms to counteract drug toxicity in some of the mutants. In essence, we describe here the first attempts to restore complete symmetry in an asymmetric ABC transporter and to study its effects, which might be relevant to the entire class of asymmetric ABC transporters.     

A Mechanism Regulating G Protein-coupled Receptor Signaling That Requires Cycles of Protein Palmitoylation and Depalmitoylation

Reversible attachment and removal of palmitate or other long-chain fatty acids on proteins has been hypothesized, like phosphorylation, to control diverse biological processes. Indeed, palmitate turnover regulates Ras trafficking and signaling. Beyond this example, however, the functions of palmitate turnover on specific proteins remain poorly understood. Here, we show that a mechanism regulating G protein-coupled receptor signaling in neuronal cells requires palmitate turnover. We used hexadecyl fluorophosphonate or palmostatin B to inhibit enzymes in the serine hydrolase family that depalmitoylate proteins, and we studied R7 regulator of G protein signaling (RGS)-binding protein (R7BP), a palmitoylated allosteric modulator of R7 RGS proteins that accelerate deactivation of G_i/o class G proteins. Depalmitoylation inhibition caused R7BP to redistribute from the plasma membrane to endomembrane compartments, dissociated R7BP-bound R7 RGS complexes from G_i/o-gated G protein-regulated inwardly rectifying K⁺ (GIRK) channels and delayed GIRK channel closure. In contrast, targeting R7BP to the plasma membrane with a polybasic domain and an irreversibly attached lipid instead of palmitate rendered GIRK channel closure insensitive to depalmitoylation inhibitors. Palmitate turnover therefore is required for localizing R7BP to the plasma membrane and facilitating G_i/o deactivation by R7 RGS proteins on GIRK channels. Our findings broaden the scope of biological processes regulated by palmitate turnover on specific target proteins. Inhibiting R7BP depalmitoylation may provide a means of enhancing GIRK activity in neurological disorders.     

Vesicle-associated Membrane Protein 2 (VAMP2) but Not VAMP3 Mediates cAMP-stimulated Trafficking of the Renal Na+-K+-2Cl? Co-transporter NKCC2 in Thick Ascending Limbs

In the kidney, epithelial cells of the thick ascending limb (TAL) reabsorb NaCl via the apical Na⁺/K⁺/2Cl⁻ co-transporter NKCC2. Steady-state surface NKCC2 levels in the apical membrane are maintained by a balance between exocytic delivery, endocytosis, and recycling. cAMP is the second messenger of hormones that enhance NaCl absorption. cAMP stimulates NKCC2 exocytic delivery via protein kinase A (PKA), increasing steady-state surface NKCC2. However, the molecular mechanism involved has not been studied. We found that several members of the SNARE family of membrane fusion proteins are expressed in TALs. Here we report that NKCC2 co-immunoprecipitates with VAMP2 in rat TALs, and they co-localize in discrete domains at the apical surface. cAMP stimulation enhanced VAMP2 exocytic delivery to the plasma membrane of renal cells, and stimulation of PKA enhanced VAMP2-NKCC2 co-immunoprecipitation in TALs. In vivo silencing of VAMP2 but not VAMP3 in TALs blunted cAMP-stimulated steady-state surface NKCC2 expression and completely blocked cAMP-stimulated NKCC2 exocytic delivery. VAMP2 was not involved in constitutive NKCC2 delivery. We concluded that VAMP2 but not VAMP3 selectively mediates cAMP-stimulated NKCC2 exocytic delivery and surface expression in TALs. We also demonstrated that cAMP stimulation enhances VAMP2 exocytosis and promotes VAMP2 interaction with NKCC2.