Aquaporin-2 in the ��-omics�� Era

Vasopressin controls renal water excretion largely through actions to regulate the water channel aquaporin-2 in collecting duct principal cells. Our knowledge of the mechanisms involved has increased markedly in recent years with the advent of methods for large-scale systems-level profiling such as protein mass spectrometry, yeast two-hybrid analysis, and oligonucleotide microarrays. Here we review this progress.Regulation of water excretion by the kidney is one of the most visible aspects of everyday physiology. An outdoor tennis game on a hot summer day can result in substantial water losses by sweating, and the kidneys respond by reducing water excretion. In contrast, excessive intake of water, a frequent occurrence in everyday life, results in excretion of copious amounts of clear urine. These responses serve to exact tight control on the tonicity of body fluids, maintaining serum osmolality in the range of 290–294 mosmol/kg of H₂O through the regulated return of water from the pro-urine in the renal collecting ducts to the bloodstream.The importance of this process is highlighted when the regulation fails. For example, polyuria (rapid uncontrolled excretion of water) is a sometimes devastating consequence of lithium therapy for bipolar disorder. On the other side of the coin are water balance disorders that result from excessive renal water retention causing systemic hypo-osmolality or hyponatremia. Hyponatremia due to excessive water retention can be seen with severe congestive heart failure, hepatic cirrhosis, and the syndrome of inappropriate antidiuresis.The chief regulator of water excretion is the peptide hormone AVP,² whereas the chief molecular target for regulation is the water channel AQP2. In this minireview, we describe new progress in the understanding of the molecular mechanisms involved in regulation of AQP2 by AVP in collecting duct cells, with emphasis on new information derived from “systems-level” approaches involving large-scale profiling and screening techniques such as oligonucleotide arrays, protein mass spectrometry, and yeast two-hybrid analysis. Most of the progress with these techniques is in the identification of individual molecules involved in AVP signaling and binding interactions with AQP2. Additional related issues are addressed in several recent reviews (1–4).     

The Role of the ��DELSEED-loop of ATP Synthase

ATP synthase uses a unique rotational mechanism to convert chemical energy into mechanical energy and back into chemical energy. The helix-turn-helix motif, termed “DELSEED-loop,” in the C-terminal domain of the β subunit was suggested to be involved in coupling between catalysis and rotation. Here, the role of the DELSEED-loop was investigated by functional analysis of mutants of Bacillus PS3 ATP synthase that had 3–7 amino acids within the loop deleted. All mutants were able to catalyze ATP hydrolysis, some at rates several times higher than the wild-type enzyme. In most cases ATP hydrolysis in membrane vesicles generated a transmembrane proton gradient, indicating that hydrolysis occurred via the normal rotational mechanism. Except for two mutants that showed low activity and low abundance in the membrane preparations, the deletion mutants were able to catalyze ATP synthesis. In general, the mutants seemed less well coupled than the wild-type enzyme, to a varying degree. Arrhenius analysis demonstrated that in the mutants fewer bonds had to be rearranged during the rate-limiting catalytic step; the extent of this effect was dependent on the size of the deletion. The results support the idea of a significant involvement of the DELSEED-loop in mechanochemical coupling in ATP synthase. In addition, for two deletion mutants it was possible to prepare an α₃β₃γ subcomplex and measure nucleotide binding to the catalytic sites. Interestingly, both mutants showed a severely reduced affinity for MgATP at the high affinity site.F₁F₀-ATP synthase catalyzes the final step of oxidative phosphorylation and photophosphorylation, the synthesis of ATP from ADP and inorganic phosphate. F₁F₀-ATP synthase consists of the membrane-embedded F₀ subcomplex, with, in most bacteria, a subunit composition of ab₂c₁₀, and the peripheral F₁ subcomplex, with a subunit composition of α₃β₃γδε. The energy necessary for ATP synthesis is derived from an electrochemical transmembrane proton (or, in some organisms, a sodium ion) gradient. Proton flow down the gradient through F₀ is coupled to ATP synthesis on F₁ by a unique rotary mechanism. The protons flow through (half) channels at the interface of the a and c subunits, which drives rotation of the ring of c subunits. The c₁₀ ring, together with F₁ subunits γ and ε, forms the rotor. Rotation of γ leads to conformational changes in the catalytic nucleotide binding sites on the β subunits, where ADP and P_i are bound. The conformational changes result in the formation and release of ATP. Thus, ATP synthase converts electrochemical energy, the proton gradient, into mechanical energy in the form of subunit rotation and back into chemical energy as ATP. In bacteria, under certain physiological conditions, the process runs in reverse. ATP is hydrolyzed to generate a transmembrane proton gradient, which the bacterium requires for such functions as nutrient import and locomotion (for reviews, see Refs. 1–6).F₁ (or F₁-ATPase) has three catalytic nucleotide binding sites located on the β subunits at the interface to the adjacent α subunit. The catalytic sites have pronounced differences in their nucleotide binding affinity. During rotational catalysis, the sites switch their affinities in a synchronized manner; the position of γ determines which catalytic site is the high affinity site (K_d1 in the nanomolar range), which site is the medium affinity site (K_d2 ≈ 1 μm), and which site is the low affinity site (K_d3 ≈ 30–100 μm; see Refs. 7 and 8). In the original crystal structure of bovine mitochondrial F₁ (9), one of the three catalytic sites, was filled with the ATP analog AMP-PNP,² a second was filled with ADP (plus azide) (see Ref. 10), and the third site was empty. Hence, the β subunits are referred to as β_TP, β_DP, and β_E. The occupied β subunits, β_TP and β_DP, were in a closed conformation, and the empty β_E subunit was in an open conformation. The main difference between these two conformations is found in the C-terminal domain. Here, the “DELSEED-loop,” a helix-turn-helix structure containing the conserved DELSEED motif, is in an “up” position when the catalytic site on the respective β subunit is filled with nucleotide and in a “down” position when the site is empty (Fig. 1A). When all three catalytic sites are occupied by nucleotide, the previously open β_E subunit assumes an intermediate, half-closed (β_HC) conformation. It cannot close completely because of steric clashes with γ (11).Open in a separate windowFIGURE 1.The βDELSEED-loop. A, interaction of the β_TP and β_E subunits with theγ subunit.β subunits are shown in yellow andγ in blue. The DELSEED-loop (shown in orange, with the DELSEED motif itself in green)of β_TP interacts with the C-terminal helixγ and the short helix that runs nearly perpendicular to the rotation axis. The DELSEED-loop of β_E makes contact with the convex portion of γ, formed mainly by the N-terminal helix. A nucleotide molecule (shown in stick representation) occupies the catalytic site of β_TP, and the subunit is in the closed conformation. The catalytic site on β_E is empty, and the subunit is in the open conformation. This figure is based on Protein Data Bank file 1e79 (32). B, deletions in the βDELSEED-loop. The loop was “mutated” in silico to represent the PS3 ATP synthase. The 3–4-residue segments that are removed in the deletion mutants are color-coded as follows: ³⁸⁰LQDI³⁸³, pink; ³⁸⁴IAIL³⁸⁷, green; ³⁸⁸GMDE³⁹¹, yellow; ³⁹²LSD³⁹⁴, cyan; ³⁹⁵EDKL³⁹⁸, orange; ³⁹⁹VVHR⁴⁰², blue. Residues that are the most involved in contacts with γ are labeled. All figures were generated using the program PyMOL (DeLano Scientific, San Carlos, CA).The DELSEED-loop of each of the three β subunits makes contact with the γ subunit. In some cases, these contacts consist of hydrogen bonds or salt bridges between the negatively charged residues of the DELSEED motif and positively charged residues on γ. The interactions of the DELSEED-loop with γ, its movement during catalysis, the conservation of the DELSEED motif (see 12–14). Thus, the finding that an AALSAAA mutant in the α₃β₃γ complex of ATP synthase from the thermophilic Bacillus PS3, where several hydrogen bonds/salt bridges to γ are removed simultaneously, could drive rotation of γ with the same torque as the wild-type enzyme (14) came as a surprise. On the other hand, it seems possible that it is the bulk of the DELSEED-loop, more so than individual interactions, that drives rotation of γ. According to a model favored by several authors (6, 15, 16) (see also Refs. 17–19), binding of ATP (or, more precisely, MgATP) to the low affinity catalytic site on β_E and the subsequent closure of this site, accompanied by its conversion into the high affinity site, are responsible for driving the large (80–90°) rotation substep during ATP hydrolysis, with the DELSEED-loop acting as a “pushrod.” A recent molecular dynamics (20) study supports this model and implicates mainly the region around several hydrophobic residues upstream of the DELSEED motif (specifically βI386 and βL387)³ as being responsible for making contact with γ during the large rotation substep.

TABLE 1

Conservation of residues in the DELSEED-loop Amino acids found in selected species in the turn region of the DELSEED-loop. Listed are all positions subjected to deletions in the present study. Residue numbers refer to the PS3 enzyme. Consensus annotation: p, polar residue; s, small residue; h, hydrophobic residue; –, negatively charged residue; +, positively charged residue.Open in a separate windowIn the present study, we investigated the function of the DELSEED-loop using an approach less focused on individual residues, by deleting stretches of 3–7 amino acids between positions β380 and β402 of ATP synthase from the thermophilic Bacillus PS3. We analyzed the functional properties of the deletion mutants after expression in Escherichia coli. The mutants showed ATPase activities, which were in some cases surprisingly high, severalfold higher than the activity of the wild-type control. On the other hand, in all cases where ATP synthesis could be measured, the rates where below or equal to those of the wild-type enzyme. In Arrhenius plots, the hydrolysis rates of the mutants were less temperature-dependent than those of wild-type ATP synthase. In those cases where nucleotide binding to the catalytic sites could be tested, the deletion mutants had a much reduced affinity for MgATP at high affinity site 1. The functional role of the DELSEED-loop will be discussed in light of the new information.     

Engineering of Ion Sensing by the Cystathionine ��-Synthase Module of the ABC Transporter OpuA

We have previously shown that the C-terminal cystathionine β-synthase (CBS) domains of the nucleotide-binding domains of the ABC transporter OpuA, in conjunction with an anionic membrane surface function, act as sensor of internal ionic strength (I_in). Here, we show that a surface-exposed cationic region in the CBS module domain is critical for ion sensing. The consecutive substitution of up to five cationic residues led to a gradual decrease of the ionic strength dependence of transport. In fact, a 5-fold mutant was essentially independent of salt in the range from 0 to 250 mm KCl (or NaCl), supplemented to medium of 30 mm potassium phosphate. Importantly, the threshold temperature for transport was lowered by 5–7 °C and the temperature coefficient Q₁₀ was lowered from 8 to ∼1.5 in the 5-fold mutant, indicating that large conformational changes are accompanying the CBS-mediated regulation of transport. Furthermore, by replacing the anionic C-terminal tail residues that extend the CBS module with histidines, the transport of OpuA became pH-dependent, presumably by additional charge interactions of the histidine residues with the membrane. The pH dependence was not observed at high ionic strength. Altogether the analyses of the CBS mutants support the notion that the osmotic regulation of OpuA involves a simple biophysical switching mechanism, in which nonspecific electrostatic interactions of a protein module with the membrane are sufficient to lock the transporter in the inactive state.In their natural habitats microorganisms are often exposed to changes in the concentration of solutes in the environment (1). A sudden increase in the medium osmolality results in loss of water from the cell, loss of turgor, a decrease in cell volume, and an increase in intracellular osmolyte concentration. Osmoregulatory transporters such as OpuA in Lactococcus lactis, ProP in Escherichia coli, and BetP in Corynebacterium glutamicum diminish the consequences of the osmotic stress by mediating the uptake of compatible solutes upon an increase in extracellular osmolality (2–4). For the ATP-binding cassette (ABC)⁵ transporter OpuA, it has been shown that the system, reconstituted in proteoliposomes, is activated by increased concentrations of lumenal ions (increased internal ionic strength) (2, 5, 6). This activation is instantaneous both in vivo and in vitro and only requires threshold levels of ionic osmolytes. Moreover, the ionic threshold for activation is highly dependent of the ionic lipid content (charge density) of the membrane and requires the presence of so-called cystathionine β-synthase (CBS) domains, suggesting that the ionic signal is transduced to the transporter via critical interactions of the protein with membrane lipids.The ABC transporter OpuA consists of two identical nucleotide-binding domains (NBD) fused to CBS domains and two identical substrate-binding domains fused to transmembrane domains. The NBD-CBS and substrate-binding domain-transmembrane domain subunits are named OpuAA and OpuABC, respectively. Two tandem CBS domains are linked to the C-terminal end of the NBD; each domain (CBS1 and CBS2) has a β-α-β-β-α secondary structure (5) (Fig. 1A). The CBS domains are widely distributed in most if not all species of life but their function is largely unknown. Most of the CBS domains are found as tandem repeats but data base searches have also revealed tetra-repeat units (5). The crystal structures of several tandem CBS domains have been elucidated (7–9, 32), and in a number of cases it has been shown that two tandem CBS domains form dimeric structures with a total of four CBS domains per structural module (hereafter referred to as CBS module). The crystal structures of the full-length MgtE Mg²⁺ transporter confirm the dimeric configuration and show that the CBS domains undergo large conformational changes upon Mg²⁺ binding or release (10, 11). In general, ABC transporters are functional as dimers, which implies that two tandem CBS domains are present in the OpuA complex. Preliminary experiments with disulfides engineered at the interface of two tandem CBS domains in OpuA suggest that large structural rearrangements (association-dissociation of the interfaces) play a determining role in the ionic strength-regulated transport. Finally, a subset of CBS-containing proteins has a C-terminal extension, which in OpuA is highly anionic (sequence: ADIPDEDEVEEIEKEEENK) and modulates the ion sensing activity (6).Open in a separate windowFIGURE 1.Domain structure of CBS module of OpuA. A, sequence of tandem CBS domains. The predicted secondary structure is indicated above the sequence. The residues modified in this study are underlined. The amino acid sequence end-points of OpuAΔ61 and OpuAΔ119 are indicated by vertical arrows. B, homology model of tandem CBS domain of OpuA. The CBS domains were individually modeled on the crystal structure of the tandem CBS protein Ta0289 from T. acidophilum (PDB entry 1PVM), using Phyre. Ta0289 was used for the initial modeling, because its primary sequence was more similar to the CBS domains of OpuA than those of the other crystallized CBS proteins. The individual domain models were then assembled with reference to the atomic coordinates of the tandem CBS domains of IMPDH from Streptococcus pyogenes (PDB entry 1ZFJ) to form the tandem CBS pair, using PyMOL (DeLano). The positions of the (substituted) cationic residues are indicated.In this study, we have engineered the surface-exposed cationic residues of the CBS module and the C-terminal anionic tail of OpuA (Fig. 1B). The ionic strength and lipid dependence of the OpuA mutants were determined in vivo and in vitro. We show that substitution of five cationic residues for neutral amino acids is sufficient to inactivate the ionic strength sensor and convert OpuA into a constitutively active transporter. Moreover, by substituting six anionic plus four neutral residues of the C-terminal anionic tail for histidines, the transport reaction becomes strongly pH-dependent.     

Molecular Basis of the Recognition of Nephronectin by Integrin ��8��1

Integrin α8β1 interacts with a variety of Arg-Gly-Asp (RGD)-containing ligands in the extracellular matrix. Here, we examined the binding activities of α8β1 integrin toward a panel of RGD-containing ligands. Integrin α8β1 bound specifically to nephronectin with an apparent dissociation constant of 0.28 ± 0.01 nm, but showed only marginal affinities for fibronectin and other RGD-containing ligands. The high-affinity binding to α8β1 integrin was fully reproduced with a recombinant nephronectin fragment derived from the RGD-containing central “linker” segment. A series of deletion mutants of the recombinant fragment identified the LFEIFEIER sequence on the C-terminal side of the RGD motif as an auxiliary site required for high-affinity binding to α8β1 integrin. Alanine scanning mutagenesis within the LFEIFEIER sequence defined the EIE sequence as a critical motif ensuring the high-affinity integrin-ligand interaction. Although a synthetic LFEIFEIER peptide failed to inhibit the binding of α8β1 integrin to nephronectin, a longer peptide containing both the RGD motif and the LFEIFEIER sequence was strongly inhibitory, and was ∼2,000-fold more potent than a peptide containing only the RGD motif. Furthermore, trans-complementation assays using recombinant fragments containing either the RGD motif or LFEIFEIER sequence revealed a clear synergism in the binding to α8β1 integrin. Taken together, these results indicate that the specific high-affinity binding of nephronectin to α8β1 integrin is achieved by bipartite interaction of the integrin with the RGD motif and LFEIFEIER sequence, with the latter serving as a synergy site that greatly potentiates the RGD-driven integrin-ligand interaction but has only marginal activity to secure the interaction by itself.Integrins are a family of adhesion receptors that interact with a variety of extracellular ligands, typically cell-adhesive proteins in the extracellular matrix (ECM).² They play mandatory roles in embryonic development and the maintenance of tissue architectures by providing essential links between cells and the ECM (1). Integrins are composed of two non-covalently associated subunits, termed α and β. In mammals, 18 α and 8 β subunits have been identified, and combinations of these subunits give rise to at least 24 distinct integrin heterodimers. Based on their ligand-binding specificities, ECM-binding integrins are classified into three groups, namely laminin-, collagen- and RGD-binding integrins (2, 3), of which the RGD-binding integrins have been most extensively investigated. The RGD-binding integrins include α5β1, α8β1, αIIbβ3, and αV-containing integrins, and have been shown to interact with a variety of ECM ligands, such as fibronectin and vitronectin, with distinct binding specificities.The α8 integrin subunit was originally identified in chick nerves (4). Integrin α8β1 is expressed in the metanephric mesenchyme and plays a crucial role in epithelial-mesenchymal interactions during the early stages of kidney morphogenesis. Disruption of the α8 gene in mice was found to be associated with severe defects in kidney morphogenesis (5) and stereocilia development (6). To date, α8β1 integrin has been shown to bind to fibronectin, vitronectin, osteopontin, latency-associated peptide of transforming growth factor-β1, tenascin-W, and nephronectin (also named POEM) (7–13), among which nephronectin is believed to be an α8β1 integrin ligand involved in kidney development (10).Nephronectin is one of the basement membrane proteins whose expression and localization patterns are restricted in a tissue-specific and developmentally regulated manner (10, 11). Nephronectin consists of five epidermal growth factor-like repeats, a linker segment containing the RGD cell-adhesive motif (designated RGD-linker) and a meprin-A5 protein-receptor protein-tyrosine phosphatase μ (MAM) domain (see Fig. 3A). Although the physiological functions of nephronectin remain only poorly understood, it is thought to play a role in epithelial-mesenchymal interactions through binding to α8β1 integrin, thereby transmitting signals from the epithelium to the mesenchyme across the basement membrane (10). Recently, mice deficient in nephronectin expression were produced by homologous recombination (14). These nephronectin-deficient mice frequently displayed kidney agenesis, a phenotype reminiscent of α8 integrin knock-out mice (14), despite the fact that other RGD-containing ligands, including fibronectin and osteopontin, were expressed in the embryonic kidneys (9, 15). The failure of the other RGD-containing ligands to compensate for the deficiency of nephronectin in the developing kidneys suggests that nephronectin is an indispensable α8β1 ligand that plays a mandatory role in epithelial-mesenchymal interactions during kidney development.Open in a separate windowFIGURE 3.Binding activities of α8β1 integrin to nephronectin and its fragments. A, schematic diagrams of full-length nephronectin (NN) and its fragments. RGD-linker and RGD-linker (GST), the central RGD-containing linker segments expressed in mammalian and bacterial expression systems, respectively; PRGDV, a short RGD-containing peptide modeled after nephronectin and expressed as a GST fusion protein (see Fig. 4A for the peptide sequence). The arrowheads indicate the positions of the RGD motif. B, purified recombinant proteins were analyzed by SDS-PAGE in 7–15% gradient (left and center panels) and 12% (right panels) gels, followed by Coomassie Brilliant Blue (CBB) staining, immunoblotting with an anti-FLAG mAb, or lectin blotting with PNA. The quantities of proteins loaded were: 0.5 μg (for Coomassie Brilliant Blue staining) and 0.1 μg (for blotting with anti-FLAG and PNA) in the left and center panels;1 μg in the right panel. C, recombinant proteins (10 nm) were coated on microtiter plates and assessed for their binding activities toward α8β1 integrin (10 nm) in the presence of 1 mm Mn²⁺. The backgrounds were subtracted as described in the legend to Fig. 2. The results represent the mean ± S.D. of triplicate determinations. D, titration curves of α8β1 integrin bound to full-length nephronectin (NN, closed squares), the RGD-linker segments expressed in 293F cells (RGD-linker, closed triangles) and E. coli (RGD-linker (GST), open triangles), the MAM domain (MAM, closed diamonds), and the PRGDV peptide expressed as a GST fusion protein in E. coli (PRGDV (GST), open circles). The assays were performed as described in the legend to Fig. 2B. The results represent the means of duplicate determinations.Although ligand recognition by RGD-binding integrins is primarily determined by the RGD motif in the ligands, it is the residues outside the RGD motif that define the binding specificities and affinities toward individual integrins (16, 17). For example, α5β1 integrin specifically binds to fibronectin among the many RGD-containing ligands, and requires not only the RGD motif in the 10th type III repeat but also the so-called “synergy site” within the preceding 9th type III repeat for fibronectin recognition (18). Recently, DiCara et al. (19) demonstrated that the high-affinity binding of αVβ6 integrin to its natural ligands, e.g. foot-and-mouth disease virus, requires the RGD motif immediately followed by a Leu-Xaa-Xaa-Leu/Ile sequence, which forms a helix to align the two conserved hydrophobic residues along the length of the helix. Given the presence of many naturally occurring RGD-containing ligands, it is conceivable that the specificities of the RGD-binding integrins are dictated by the sequences flanking the RGD motif or those in neighboring domains that come into close proximity with the RGD motif in the intact ligand proteins. However, the preferences of α8β1 integrin for RGD-containing ligands and how it secures its high-affinity binding toward its preferred ligands remain unknown.In the present study, we investigated the binding specificities of α8β1 integrin toward a panel of RGD-containing cell-adhesive proteins. Our data reveal that nephronectin is a preferred ligand for α8β1 integrin, and that a LFEIFEIER sequence on the C-terminal side of its RGD motif serves as a synergy site to ensure the specific high-affinity binding of nephronectin to α8β1 integrin.     

An N-Glycosylation Site on the��-Propeller Domain of the Integrin ��5 Subunit Plays Key Roles in Both Its Function and Site-specific Modification by��1,4-N-Acetylglucosaminyltransferase III

Recently we reported that N-glycans on the β-propeller domain of the integrin α5 subunit (S-3,4,5) are essential for α5β1 heterodimerization, expression, and cell adhesion. Herein to further investigate which N-glycosylation site is the most important for the biological function and regulation, we characterized the S-3,4,5 mutants in detail. We found that site-4 is a key site that can be specifically modified by N-acetylglucosaminyltransferase III (GnT-III). The introduction of bisecting GlcNAc into the S-3,4,5 mutant catalyzed by GnT-III decreased cell adhesion and migration on fibronectin, whereas overexpression of N-acetylglucosaminyltransferase V (GnT-V) promoted cell migration. The phenomenon is similar to previous observations that the functions of the wild-type α5 subunit were positively and negatively regulated by GnT-V and GnT-III, respectively, suggesting that the α5 subunit could be duplicated by the S-3,4,5 mutant. Interestingly GnT-III specifically modified the S-4,5 mutant but not the S-3,5 mutant. This result was confirmed by erythroagglutinating phytohemagglutinin lectin blot analysis. The reduction in cell adhesion was consistently observed in the S-4,5 mutant but not in the S-3,5 mutant cells. Furthermore mutation of site-4 alone resulted in a substantial decrease in erythroagglutinating phytohemagglutinin lectin staining and suppression of cell spread induced by GnT-III compared with that of either the site-3 single mutant or wild-type α5. These results, taken together, strongly suggest that N-glycosylation of site-4 on the α5 subunit is the most important site for its biological functions. To our knowledge, this is the first demonstration that site-specific modification of N-glycans by a glycosyltransferase results in functional regulation.Glycosylation is a crucial post-translational modification of most secreted and cell surface proteins (1). Glycosylation is involved in a variety of physiological and pathological events, including cell growth, migration, differentiation, and tumor invasion. It is well known that glycans play important roles in cell-cell communication, intracellular signal transduction, protein folding, and stability (2, 3).Integrins comprise a family of receptors that are important for cell adhesion. The major function of integrins is to connect cells to the extracellular matrix, activate intracellular signaling pathways, and regulate cytoskeletal formation (4). Integrin α5β1 is well known as a fibronectin (FN)³ receptor. The interaction between integrin α5 and FN is essential for cell migration, cell survival, and development (5–8). In addition, integrins are N-glycan carrier proteins. For example, α5β1 integrin contains 14 and 12 putative N-glycosylation sites on the α5 and β1 subunits, respectively. Several studies suggest that N-glycosylation is essential for functional integrin α5β1. When human fibroblasts were cultured in the presence of 1-deoxymannojirimycin, which prevents N-linked oligosaccharide processing, immature α5β1 integrin appeared on the cell surface, and FN-dependent adhesion was greatly reduced (9). Treatment of purified integrin α5β1 with N-glycosidase F, which cleaves between the innermost N-acetylglucosamine (GlcNAc) and asparagine N-glycan residues of N-linked glycoproteins, prevented the inherent association between subunits and blocked α5β1 binding to FN (10).A growing body of evidence indicates that the presence of the appropriate oligosaccharide can modulate integrin activation. N-Acetylglucosaminyltransferase III (GnT-III) catalyzes the addition of GlcNAc to mannose that is β1,4-linked to an underlying N-acetylglucosamine, producing what is known as a “bisecting” GlcNAc linkage as shown in Fig. 1B. GnT-III is generally regarded as a key glycosyltransferase in N-glycan biosynthetic pathways and contributes to inhibition of metastasis. The introduction of a bisecting GlcNAc catalyzed by GnT-III suppresses additional processing and elongation of N-glycans. These reactions, which are catalyzed in vitro by other glycosyltransferases, such as N-acetylglucosaminyltransferase V (GnT-V), which catalyzes the formation of β1,6 GlcNAc branching structures (Fig. 1B) and plays important roles in tumor metastasis, do not proceed because the enzymes cannot utilize the bisected N-glycans as a substrate. Introduction of the bisecting GlcNAc to integrin α5 by overexpression of GnT-III resulted in decreased in ligand binding and down-regulation of cell adhesion and migration (11–13). Contrary to the functions of GnT-III, overexpression of GnT-V promoted integrin α5β1-mediated cell migration on FN (14). These observations clearly demonstrate that the alteration of N-glycan structure affected the biological functions of integrin α5β1. Similarly characterization of the carbohydrate moieties in integrin α3β1 from non-metastatic and metastatic human melanoma cell lines showed that expression of β1,6 GlcNAc branched structures was higher in metastatic cells compared with non-metastatic cells, confirming the notion that the β1,6 GlcNAc branched structure confers invasive and metastatic properties to cancer cells. In fact, Partridge et al. (15) reported that GnT-V-modified N-glycans containing poly-N-acetyllactosamine, the preferred ligand for galectin-3, on surface receptors oppose their constitutive endocytosis, promoting intracellular signaling and consequently cell migration and tumor metastasis.Open in a separate windowFIGURE 1.Potential N-glycosylation sites on the α5 subunit and its modification by GnT-III and GnT-V. A, schematic diagram of potential N-glycosylation sites on the α5 subunit. Putative N-glycosylation sites are indicated by triangles, and point mutations are indicated by crosses (N84Q, N182Q, N297Q, N307Q, N316Q, N524Q, N530Q, N593Q, N609Q, N675Q, N712Q, N724Q, N773Q, and N868Q). B, illustration of the reaction catalyzed by GnT-III and GnT-V. Square, GlcNAc; circle, mannose. TM, transmembrane domain.In addition, sialylation on the non-reducing terminus of N-glycans of α5β1 integrin plays an important role in cell adhesion. Colon adenocarcinomas express elevated levels of α2,6 sialylation and increased activity of ST6GalI sialyltransferase. Elevated ST6GalI positively correlated with metastasis and poor survival. Therefore, ST6GalI-mediated hypersialylation likely plays a role in colorectal tumor invasion (16, 17). In fact, oncogenic ras up-regulated ST6GalI and, in turn, increased sialylation of β1 integrin adhesion receptors in colon epithelial cells (18). However, this is not always the case. The expression of hyposialylated integrin α5β1 was induced by phorbol esterstimulated differentiation in myeloid cells in which the expression of the ST6GalI was down-regulated by the treatment, increasing FN binding (19). A similar phenomenon was also observed in hematopoietic or other epithelial cells. In these cells, the increased sialylation of the β1 integrin subunit was correlated with reduced adhesiveness and metastatic potential (20–22). In contrast, the enzymatic removal of α2,8-linked oligosialic acids from the α5 integrin subunit inhibited cell adhesion to FN (23). Collectively these findings suggest that the interaction of integrin α5β1 with FN is dependent on its N-glycosylation and the processing status of N-glycans.Because integrin α5β1 contains multipotential N-glycosylation sites, it is important to determine the sites that are crucial for its biological function and regulation. Recently we found that N-glycans on the β-propeller domain (sites 3, 4, and 5) of the integrin α5 subunit are essential for α5β1 heterodimerization, cell surface expression, and biological function (24). In this study, to further investigate the underlying molecular mechanism of GnT-III-regulated biological functions, we characterized the N-glycans on the α5 subunit in detail using genetic and biochemical approaches and found that site-4 is a key site that can be specifically modified by GnT-III.     

Involvement of Acid ��-Glucosidase 1 in the Salvage Pathway of Ceramide Formation

Activation of protein kinase C (PKC) promotes the salvage pathway of ceramide formation, and acid sphingomyelinase has been implicated, in part, in providing substrate for this pathway (Zeidan, Y. H., and Hannun, Y. A. (2007) J. Biol. Chem. 282, 11549–11561). In the present study, we examined whether acid β-glucosidase 1 (GBA1), which hydrolyzes glucosylceramide to form lysosomal ceramide, was involved in PKC-regulated formation of ceramide from recycled sphingosine. Glucosylceramide levels declined after treatment of MCF-7 cells with a potent PKC activator, phorbol 12-myristate 13-acetate (PMA). Silencing GBA1 by small interfering RNAs significantly attenuated acid glucocerebrosidase activity and decreased PMA-induced formation of ceramide by 50%. Silencing GBA1 blocked PMA-induced degradation of glucosylceramide and generation of sphingosine, the source for ceramide biosynthesis. Reciprocally, forced expression of GBA1 increased ceramide levels. These observations indicate that GBA1 activation can generate the source (sphingosine) for PMA-induced formation of ceramide through the salvage pathway. Next, the role of PKCδ, a direct effector of PMA, in the formation of ceramide was determined. By attenuating expression of PKCδ, cells failed to trigger PMA-induced alterations in levels of ceramide, sphingomyelin, and glucosylceramide. Thus, PKCδ activation is suggested to stimulate the degradation of both sphingomyelin and glucosylceramide leading to the salvage pathway of ceramide formation. Collectively, GBA1 is identified as a novel source of regulated formation of ceramide, and PKCδ is an upstream regulator of this pathway.Sphingolipids are abundant components of cellular membranes, many of which are emerging as bioactive lipid mediators thought to play crucial roles in cellular responses (1, 2). Ceramide, a central sphingolipid, serves as the main precursor for various sphingolipids, including glycosphingolipids, gangliosides, and sphingomyelin. Regulation of formation of ceramide has been demonstrated through the action of three major pathways: the de novo pathway (3, 4), the sphingomyelinase pathway (5), and the salvage pathway (6–8). The latter plays an important role in constitutive sphingolipid turnover by salvaging long-chain sphingoid bases (sphingosine and dihydrosphingosine) that serve as sphingolipid backbones for ceramide and dihydroceramide as well as all complex sphingolipids (Fig. 1A).Open in a separate windowFIGURE 1.The scheme of the sphingosine salvage pathway of ceramide formation and inhibition of PMA induction of ceramide by fumonisin B1. A, the scheme of the sphingosine salvage pathway of ceramide formation. B, previously published data as to effects of fumonisin B1 on ceramide mass profiles (23) are re-plotted as a PMA induction of ceramide. In brief, MCF-7 cells were pretreated with or without 100 μm fumonisin B1 for 2 h followed by treatment with 100 nm PMA for 1 h. Lipids were extracted, and then the levels of ceramide species were determined by high-performance liquid chromatography-tandem mass spectrometry. Results are expressed as sum of increased mass of ceramide species. Dotted or open columns represents C₁₆-ceramide or sum of other ceramide species (C₁₄-ceramide, C₁₈-ceramide, C_18:1-ceramide, C₂₀-ceramide, C₂₄-ceramide, and C_24:1-ceramide), respectively. The data represent mean ± S.E. of three to five values.Metabolically, ceramide is also formed from degradation of glycosphingolipids (Fig. 1A) usually in acidic compartments, the lysosomes and/or late endosomes (9). The stepwise hydrolysis of complex glycosphingolipids eventually results in the formation of glucosylceramide, which in turn is converted to ceramide by the action of acid β-glucosidase 1 (GBA1)² (9, 10). Severe defects in GBA1 activity cause Gaucher disease, which is associated with aberrant accumulation of the lipid substrates (10–14). On the other hand, sphingomyelin is cleaved by acid sphingomyelinase to also form ceramide (15, 16). Either process results in the generation of lysosomal ceramide that can then be deacylated by acid ceramidase (17), releasing sphingosine that may escape the lysosome (18). The released sphingosine may become a substrate for either sphingosine kinases or ceramide synthases, forming sphingosine 1-phosphate or ceramide, respectively (3, 19–21).In a related line of investigation, our studies (20, 22, 23) have begun to implicate protein kinase Cs (PKC) as upstream regulators of the sphingoid base salvage pathway resulting in ceramide synthesis. Activation of PKCs by the phorbol ester (PMA) was shown to stimulate the salvage pathway resulting in increases in ceramide. All the induced ceramide was inhibited by pretreatment with a ceramide synthase inhibitor, fumonisin B1, but not by myriocin, thus negating acute activation of the de novo pathway and establishing a role for ceramide synthesis (20, 23). Moreover, labeling studies also implicated the salvage pathway because PMA induced turnover of steady state-labeled sphingolipids but did not affect de novo labeled ceramide in pulse-chase experiments.Moreover, PKCδ, among PKC isoforms, was identified as an upstream molecule for the activation of acid sphingomyelinase in the salvage pathway (22). Interestingly, the PKCδ isoform induced the phosphorylation of acid sphingomyelinase at serine 508, leading to its activation and consequent formation of ceramide. The activation of acid sphingomyelinase appeared to contribute to ∼50% of the salvage pathway-induced increase in ceramide (28) (also, see Fig. 4C). This raised the possibility that distinct routes of ceramide metabolism may account for the remainder of ceramide generation. In this study, we investigated glucocerebrosidase GBA1 as a candidate for one of the other routes accounting for PKC-regulated salvage pathway of ceramide formation.Open in a separate windowFIGURE 4.Effects of knockdown of lysosomal enzymes on the generation of ceramide after PMA treatment. A, MCF-7 cells were transfected with 5 nm siRNAs of each of four individual sequences (SCR, GBA1-a, GBA1-b, and GBA1-c) for 48 h and then stimulated with 100 nm PMA for 1 h. Lipids were extracted, and then the levels of the C₁₆-ceramide species were determined by high-performance liquid chromatography-tandem mass spectrometry. The data represent mean ± S.E. of three to nine values. B, MCF-7 cells were transfected with 5 nm siRNAs of SCR or GBA1-a (GBA1) for 48 h and then stimulated with 100 nm PMA for 1 h. Lipids were extracted, and then the levels of individual ceramide species were determined by high-performance liquid chromatography-tandem mass spectrometry. The data represent mean ± S.E. of three to five values. C₁₄-Cer, C₁₄-ceramide; C₁₆-Cer, C₁₆-ceramide; C₁₈-Cer; C₁₈-ceramide; C_18:1-Cer, C_18:1-ceramide; C₂₀-Cer, C₂₀-ceramide; C₂₀-Cer, C₂₄-ceramide; C_24:1-Cer, C_24:1-ceramide. C, MCF-7 cells were transfected with 5 nm siRNAs of SCR, acid sphingomyelinase (ASM), or GBA1-a (GBA1) for 48 h following stimulation with (PMA) or without (Control) 100 nm PMA for 1 h. Lipids were extracted, and then the levels of ceramide species were determined by high-performance liquid chromatography-tandem mass spectrometry. Levels of C₁₆-ceramide are shown. The data represent mean ± S.E. of four to five values. Significant changes from SCR-transfected cells treated with PMA are shown in A–C (^*, p < 0.02; ^**, p < 0.05; ^***, p < 0.01).     

CP12 from Chlamydomonas reinhardtii, a Permanent Specific ��Chaperone-like�� Protein of Glyceraldehyde-3-phosphate Dehydrogenase

A new role is reported for CP12, a highly unfolded and flexible protein, mainly known for its redox function with A₄ glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both reduced and oxidized CP12 can prevent the in vitro thermal inactivation and aggregation of GAPDH from Chlamydomonas reinhardtii. This mechanism is thus not redox-dependent. The protection is specific to CP12, because other proteins, such as bovine serum albumin, thioredoxin, and a general chaperone, Hsp33, do not fully prevent denaturation of GAPDH. Furthermore, CP12 acts as a specific chaperone, since it does not protect other proteins, such as catalase, alcohol dehydrogenase, or lysozyme. The interaction between CP12 and GAPDH is necessary to prevent the aggregation and inactivation, since the mutant C66S that does not form any complex with GAPDH cannot accomplish this protection. Unlike the C66S mutant, the C23S mutant that lacks the N-terminal bridge is partially able to protect and to slow down the inactivation and aggregation. Tryptic digestion coupled to mass spectrometry confirmed that the S-loop of GAPDH is the interaction site with CP12. Thus, CP12 not only has a redox function but also behaves as a specific “chaperone-like protein” for GAPDH, although a stable and not transitory interaction is observed. This new function of CP12 may explain why it is also present in complexes involving A₂B₂ GAPDHs that possess a regulatory C-terminal extension (GapB subunit) and therefore do not require CP12 to be redox-regulated.CP12 is a small 8.2-kDa protein present in the chloroplasts of most photosynthetic organisms, including cyanobacteria (1, 2), higher plants (3), the diatom Asterionella formosa (4, 5), and green (1) and red algae (6). It allows the formation of a supramolecular complex between phosphoribulokinase (EC 2.7.1.19) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH),³ two key enzymes of the Calvin cycle pathway, and was recently shown to interact with fructose bisphosphate aldolase, another enzyme of the Calvin cycle pathway (7). The phosphoribulokinase·GAPDH·CP12 complex has been extensively studied in Chlamydomonas reinhardtii (8, 9) and in Arabidopsis thaliana (10, 11). In the green alga C. reinhardtii, the interaction between CP12 and GAPDH is strong (8). GAPDH may exist as a homotetramer composed of four GapA subunits (A₄) in higher plants, cyanobacteria, and green and red algae (6, 12), but in higher plants, it can also exist as a heterotetramer (A₂B₂), composed of two subunits, GapA and GapB (13, 14). GapB, up to now, has exclusively been found in Streptophyta, but recently two prasinophycean green algae, Ostreococcus tauri and Ostreococcus lucimarinus, were also shown to possess a GapB gene, whereas CP12 is missing (15). The GapB subunit is similar to the GapA subunit but has a C-terminal extension containing two redox-regulated cysteine residues (16). Thus, although the A₄ GAPDHs lack these regulatory cysteine residues (13, 14, 17–20), they are also redox-regulated through its interaction with CP12, since the C terminus of this small protein resembles the C-terminal extension of the GapB subunit. The regulatory cysteine residues for GapA are thus supplied by CP12, as is well documented in the literature (1, 8, 11, 16).CP12 belongs to the family of intrinsically unstructured proteins (IUPs) (21–26). The amino acid composition of these proteins causes them to have no or few secondary structures. Their total or partial lack of structure and their high flexibility allow them to be molecular adaptors (27, 28). They are often able to bind to several partners and are involved in most cellular functions (29, 30). Recently, some IUPs have been described in photosynthetic organisms (31, 32).There are many functional categories of IUPs (22, 33). They can be, for instance, involved in permanent binding and have (i) a scavenger role, neutralizing or storing small ligands; (ii) an assembler role by forming complexes; and (iii) an effector role by modulating the activity of a partner molecule (33). These functions are not exclusive; thus, CP12 can form a stable complex with GAPDH, regulating its redox properties (8, 34, 35), and can also bind a metal ion (36, 37). IUPs can also bind transiently to partners, and some of them have been found to possess a chaperone activity (31, 38). This chaperone function was first shown for α-synuclein (39) and for α-casein (40), which are fully disordered. The amino acid composition of IUPs is less hydrophobic than those of soluble proteins; hence, they lack hydrophobic cores and do not become insoluble when heated. Since CP12 belongs to this family, we tested if it was resistant to heat treatment and finally, since it is tightly bound to GAPDH, if it could prevent aggregation of its partner, GAPDH, an enzyme well known for its tendency to aggregate (41–44) and consequently a substrate commonly used in chaperone studies (45, 46).Unlike chaperones, which form transient, dynamic complexes with their protein substrates through hydrophobic interactions (47, 48), CP12 forms a stable complex with GAPDH. The interaction involves the C-terminal part of the protein and the presence of negatively charged residues on CP12 (35). However, only a site-directed mutagenesis has been performed to characterize the interaction site on GAPDH. Although the mutation could have an indirect effect, the residue Arg-197 was shown to be a good candidate for the interaction site (49).In this report, we accordingly used proteolysis experiments coupled with mass spectrometry to detect which regions of GAPDH are protected by its association with CP12. To conclude, the aim of this report was to characterize a chaperone function of CP12 that had never been described before and to map the interaction site on GAPDH using an approach that does not involve site-directed mutagenesis.     

Catalytic Role of the Metal Ion in the Metallo-��-lactamase GOB

Metallo-β-lactamases (MβLs) stand as one of the main mechanisms of bacterial resistance toward carbapenems. The rational design of an inhibitor for MβLs has been limited by an incomplete knowledge of their catalytic mechanism and by the structural diversity of their active sites. Here we show that the MβL GOB from Elizabethkingia meningoseptica is active as a monometallic enzyme by using different divalent transition metal ions as surrogates of the native Zn(II) ion. Of the metal derivatives in which Zn(II) is replaced, Co(II) and Cd(II) give rise to the most active enzymes and are shown to occupy the same binding site as the native ion. However, Zn(II) is the only metal ion capable of stabilizing an anionic intermediate that accumulates during nitrocefin hydrolysis, in which the C–N bond has already been cleaved. This finding demonstrates that the catalytic role of the metal ion in GOB is to stabilize the formation of this intermediate prior to nitrogen protonation. This role may be general to all MβLs, whereas nucleophile activation by a Zn(II) ion is not a conserved mechanistic feature.     

N-Glycosylation of the I-like Domain of ��1 Integrin Is Essential for ��1 Integrin Expression and Biological Function: IDENTIFICATION OF THE MINIMAL N-GLYCOSYLATION REQUIREMENT FOR ��5��1*

N-Glycosylation of integrin α5β1 plays a crucial role in cell spreading, cell migration, ligand binding, and dimer formation, but the detailed mechanisms by which N-glycosylation mediates these functions remain unclear. In a previous study, we showed that three potential N-glycosylation sites (α5S3–5) on the β-propeller of the α5 subunit are essential to the functional expression of the subunit. In particular, site 5 (α5S5) is the most important for its expression on the cell surface. In this study, the function of the N-glycans on the integrin β1 subunit was investigated using sequential site-directed mutagenesis to remove the combined putative N-glycosylation sites. Removal of the N-glycosylation sites on the I-like domain of the β1 subunit (i.e. the Δ4-6 mutant) decreased both the level of expression and heterodimeric formation, resulting in inhibition of cell spreading. Interestingly, cell spreading was observed only when the β1 subunit possessed these three N-glycosylation sites (i.e. the S4-6 mutant). Furthermore, the S4-6 mutant could form heterodimers with either α5S3-5 or α5S5 mutant of the α5 subunit. Taken together, the results of the present study reveal for the first time that N-glycosylation of the I-like domain of the β1 subunit is essential to both the heterodimer formation and biological function of the subunit. Moreover, because the α5S3-5/β1S4-6 mutant represents the minimal N-glycosylation required for functional expression of the β1 subunit, it might also be useful for the study of molecular structures.Integrin is a heterodimeric glycoprotein that consists of both an α and a β subunit (1). The interaction between integrin and the extracellular matrix is essential to both physiologic and pathologic events, such as cell migration, development, cell viability, immune homeostasis, and tumorigenesis (2, 3). Among the integrin superfamily, β1 integrin can combine with 12 distinct α subunits (α1–11, αv) to form heterodimers, thereby acquiring a wide variety of ligand specificity (1, 4). Integrins are thought to be regulated by inside-out signaling mechanisms that provoke conformational changes, which modulate the affinity of integrin for the ligand (5). However, an increasing body of evidence suggests that cell-surface carbohydrates mediate a variety of interactions between integrin and its extracellular environment, thereby affecting integrin activity and possibly tumor metastasis as well (6–8).Guo et al. (9) reported that an increase in β1–6-GlcNAc sugar chains on the integrin β1 subunit stimulated cell migration. In addition, elevated sialylation of the β1 subunit, because of Ras-induced STGal-I transferase activity, also induced cell migration (10, 11). Conversely, cell migration and spreading were reduced by the addition of a bisecting GlcNAc, which is a product of N-acetylglucosaminyltransferase III (GnT-III),² to the α5β1 and α3β1 integrins (12, 13). Alterations of N-glycans on integrins might also regulate their cis interactions with membrane-associated proteins, including the epidermal growth factor receptor, the galectin family, and the tetraspanin family of proteins (14–19).In addition to the positive and negative regulatory effects of N-glycan, several research groups have reported that N-glycans must be present on integrin α5β1 for the αβ heterodimer formation and proper integrin-matrix interactions. Consistent with this hypothesis, in the presence of the glycosylation inhibitor, tunicamycin, normal integrin-substrate binding and transport to the cell surface are inhibited (20). Moreover, treatment of purified integrin with N-glycosidase F blocked both the inherent association of the subunits and the interaction between integrin and fibronectin (FN) (21). These results suggest that N-glycosylation is essential to the functional expression of α5β1. However, because integrin α5β1 contains 26 potential N-linked glycosylation sites, 14 in the α subunit and 12 in the β subunit, identification of the sites that are essential to its biological functions is key to understanding the molecular mechanisms by which N-glycans alter integrin function. Recently, our group determined that N-glycosylation of the β-propeller domain on the α5 subunit is essential to both heterodimerization and biological functions of the subunit. Furthermore, we determined that sites 3–5 are the most important sites for α5 subunit-mediated cell spreading and migration on FN (22). The purpose of this study was to clarify the roles of N-glycosylation of the β1 subunit. Therefore, we performed combined substitutions in the putative N-glycosylation sites by replacement of asparagine residues with glutamine residues. We subsequently introduced these mutated genes into β1-deficient epithelial cells (GE11). The results of these mutation experiments revealed that the N-glycosylation sites on the I-like domain of the β1 subunit, sites number 4–6 (S4-6), are essential to both heterodimer formation and biological functions, such as cell spreading.     

Functional Ryanodine Receptors in the Plasma Membrane of RINm5F Pancreatic ��-Cells

Ryanodine receptors (RyR) are Ca²⁺ channels that mediate Ca²⁺ release from intracellular stores in response to diverse intracellular signals. In RINm5F insulinoma cells, caffeine, and 4-chloro-m-cresol (4CmC), agonists of RyR, stimulated Ca²⁺ entry that was independent of store-operated Ca²⁺ entry, and blocked by prior incubation with a concentration of ryanodine that inactivates RyR. Patch-clamp recording identified small numbers of large-conductance (γ_K = 169 pS) cation channels that were activated by caffeine, 4CmC or low concentrations of ryanodine. Similar channels were detected in rat pancreatic β-cells. In RINm5F cells, the channels were blocked by cytosolic, but not extracellular, ruthenium red. Subcellular fractionation showed that type 3 IP₃ receptors (IP₃R3) were expressed predominantly in endoplasmic reticulum, whereas RyR2 were present also in plasma membrane fractions. Using RNAi selectively to reduce expression of RyR1, RyR2, or IP₃R3, we showed that RyR2 mediates both the Ca²⁺ entry and the plasma membrane currents evoked by agonists of RyR. We conclude that small numbers of RyR2 are selectively expressed in the plasma membrane of RINm5F pancreatic β-cells, where they mediate Ca²⁺ entry.Ryanodine receptors (RyR)³ and inositol 1,4,5-trisphosphate receptors (IP₃R) (1, 2) are the archetypal intracellular Ca²⁺ channels. Both are widely expressed, although RyR are more restricted in their expression than IP₃R (3, 4). In common with many cells, pancreatic β-cells and insulin-secreting cell lines express both IP₃R (predominantly IP₃R3) (5, 6) and RyR (predominantly RyR2) (7). Both RyR and IP₃R are expressed mostly within membranes of the endoplasmic (ER), where they mediate release of Ca²⁺. Functional RyR are also expressed in the secretory vesicles (8, 9) or, and perhaps more likely, in the endosomes of β-cells (10). Despite earlier suggestions (11), IP₃R are probably not present in the secretory vesicles of β-cells (8, 12, 13).All three subtypes of IP₃R are stimulated by IP₃ with Ca²⁺ (1), and the three subtypes of RyR are each directly regulated by Ca²⁺. However, RyR differ in whether their most important physiological stimulus is depolarization of the plasma membrane (RyR1), Ca²⁺ (RyR2) or additional intracellular messengers like cyclic ADP-ribose. The latter stimulates both Ca²⁺ release and insulin secretion in β-cells (8, 14). The activities of both families of intracellular Ca²⁺ channels are also modulated by many additional signals that act directly or via phosphorylation (15, 16). Although they commonly mediate release of Ca²⁺ from the ER, both IP₃R and RyR select rather poorly between Ca²⁺ and other cations (permeability ratio, P_Ca/P_K ∼7) (1, 17). This may allow electrogenic Ca²⁺ release from the ER to be rapidly compensated by uptake of K⁺ (18), and where RyR or IP₃R are expressed in other membranes it may allow them to affect membrane potential.Both Ca²⁺ entry and release of Ca²⁺ from intracellular stores contribute to the oscillatory increases in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) that stimulate exocytosis of insulin-containing vesicles in pancreatic β-cells (7). Glucose rapidly equilibrates across the plasma membrane (PM) of β-cells and its oxidative metabolism by mitochondria increases the cytosolic ATP/ADP ratio, causing K_ATP channels to close (19). This allows an unidentified leak current to depolarize the PM (20) and activate voltage-gated Ca²⁺ channels, predominantly L-type Ca²⁺ channels (21). The resulting Ca²⁺ entry is amplified by Ca²⁺-induced Ca²⁺ release from intracellular stores (7), triggering exocytotic release of insulin-containing dense-core vesicles (22). The importance of this sequence is clear from the widespread use of sulfonylurea drugs, which close K_ATP channels, in the treatment of type 2 diabetes. Ca²⁺ uptake by mitochondria beneath the PM further stimulates ATP production, amplifying the initial response to glucose and perhaps thereby contributing to the sustained phase of insulin release (23). However, neither the increase in [Ca²⁺]_i nor the insulin release evoked by glucose or other nutrients is entirely dependent on Ca²⁺ entry (7, 24) or closure of K_ATP channels (25). This suggests that glucose metabolism may also more directly activate RyR (7, 26) and/or IP₃R (27) to cause release of Ca²⁺ from intracellular stores. A change in the ATP/ADP ratio is one means whereby nutrient metabolism may be linked to opening of intracellular Ca²⁺ channels because both RyR (28) and IP₃R (1) are stimulated by ATP.The other major physiological regulators of insulin release are the incretins: glucagon-like peptide-1 and glucose-dependent insulinotropic hormone (29). These hormones, released by cells in the small intestine, stimulate synthesis of cAMP in β-cells and thereby potentiate glucose-evoked insulin release (30). These pathways are also targets of drugs used successfully to treat type 2 diabetes (29). The responses of β-cells to cAMP involve both cAMP-dependent protein kinase and epacs (exchange factors activated by cAMP) (31, 32). The effects of the latter are, at least partly, due to release of Ca²⁺ from intracellular stores via RyR (33–35) and perhaps also via IP₃R (36). The interplays between Ca²⁺ and cAMP signaling generate oscillatory changes in the concentrations of both messengers (37). RyR and IP₃R are thus implicated in mediating responses to each of the major physiological regulators of insulin secretion: glucose and incretins.Here we report that in addition to expression in intracellular stores, which probably include both the ER and secretory vesicles and/or endosomes, functional RyR2 are also expressed in small numbers in the PM of RINm5F insulinoma cells and rat pancreatic β-cells.     

Proper Restoration of Excitation-Contraction Coupling in the Dihydropyridine Receptor ��1-null Zebrafish Relaxed Is an Exclusive Function of the ��1a Subunit

The paralyzed zebrafish strain relaxed carries a null mutation for the skeletal muscle dihydropyridine receptor (DHPR) β_1a subunit. Lack of β_1a results in (i) reduced membrane expression of the pore forming DHPR α_1S subunit, (ii) elimination of α_1S charge movement, and (iii) impediment of arrangement of the DHPRs in groups of four (tetrads) opposing the ryanodine receptor (RyR1), a structural prerequisite for skeletal muscle-type excitation-contraction (EC) coupling. In this study we used relaxed larvae and isolated myotubes as expression systems to discriminate specific functions of β_1a from rather general functions of β isoforms. Zebrafish and mammalian β_1a subunits quantitatively restored α_1S triad targeting and charge movement as well as intracellular Ca²⁺ release, allowed arrangement of DHPRs in tetrads, and most strikingly recovered a fully motile phenotype in relaxed larvae. Interestingly, the cardiac/neuronal β_2a as the phylogenetically closest, and the ancestral housefly β_M as the most distant isoform to β_1a also completely recovered α_1S triad expression and charge movement. However, both revealed drastically impaired intracellular Ca²⁺ transients and very limited tetrad formation compared with β_1a. Consequently, larval motility was either only partially restored (β_2a-injected larvae) or not restored at all (β_M). Thus, our results indicate that triad expression and facilitation of 1,4-dihydropyridine receptor (DHPR) charge movement are common features of all tested β subunits, whereas the efficient arrangement of DHPRs in tetrads and thus intact DHPR-RyR1 coupling is only promoted by the β_1a isoform. Consequently, we postulate a model that presents β_1a as an allosteric modifier of α_1S conformation enabling skeletal muscle-type EC coupling.Excitation-contraction (EC)³ coupling in skeletal muscle is critically dependent on the close interaction of two distinct Ca²⁺ channels. Membrane depolarizations of the myotube are sensed by the voltage-dependent 1,4-dihydropyridine receptor (DHPR) in the sarcolemma, leading to a rearrangement of charged amino acids (charge movement) in the transmembrane segments S4 of the pore-forming DHPR α_1S subunit (1, 2). This conformational change induces via protein-protein interaction (3, 4) the opening of the sarcoplasmic type-1 ryanodine receptor (RyR1) without need of Ca²⁺ influx through the DHPR (5). The release of Ca²⁺ from the sarcoplasmic reticulum via RyR1 consequently induces muscle contraction. The protein-protein interaction mechanism between DHPR and RyR1 requires correct ultrastructural targeting of both channels. In Ca²⁺ release units (triads and peripheral couplings) of the skeletal muscle, groups of four DHPRs (tetrads) are coupled to every other RyR1 and hence are geometrically arranged following the RyR-specific orthogonal arrays (6).The skeletal muscle DHPR is a heteromultimeric protein complex, composed of the voltage-sensing and pore-forming α_1S subunit and auxiliary subunits β_1a, α₂δ-1, and γ₁ (7). While gene knock-out of the DHPR γ₁ subunit (8, 9) and small interfering RNA knockdown of the DHPR α₂δ-1 subunit (10-12) have indicated that neither subunit is essential for coupling of the DHPR with RyR1, the lack of the α_1S or of the intracellular β_1a subunit is incompatible with EC coupling and accordingly null model mice die perinatally due to asphyxia (13, 14). β subunits of voltage-gated Ca²⁺ channels were repeatedly shown to be responsible for the facilitation of α₁ membrane insertion and to be potent modulators of α₁ current kinetics and voltage dependence (15, 16). Whether the loss of EC coupling in β₁-null mice was caused by decreased DHPR membrane expression or by the lack of a putative specific contribution of the β subunit to the skeletal muscle EC coupling apparatus (17, 18) was not clearly resolved. Recently, other β-functions were identified in skeletal muscle using the β₁-null mutant zebrafish relaxed (19, 20). Like the β₁-knock-out mouse (14) zebrafish relaxed is characterized by complete paralysis of skeletal muscle (21, 22). While β₁-knock-out mouse pups die immediately after birth due to respiratory paralysis (14), larvae of relaxed are able to survive for several days because of oxygen and metabolite diffusion via the skin (23). Using highly differentiated myotubes that are easy to isolate from these larvae, the lack of EC coupling could be described by quantitative immunocytochemistry as a moderate ∼50% reduction of α_1S membrane expression although α_1S charge movement was nearly absent, and, most strikingly, as the complete lack of the arrangement of DHPRs in tetrads (19). Thus, in skeletal muscle the β subunit enables EC coupling by (i) enhancing α_1S membrane targeting, (ii) facilitating α_1S charge movement, and (iii) enabling the ultrastructural arrangement of DHPRs in tetrads.The question arises, which of these functions are specific for the skeletal muscle β_1a and which ones are rather general properties of Ca²⁺ channel β subunits. Previous reconstitution studies made in the β₁-null mouse system (24, 25) using different β subunit constructs (26) did not allow differentiation between β-induced enhancement of non-functional α_1S membrane expression and the facilitation of α_1S charge movement, due to the lack of information on α_1S triad expression levels. Furthermore, the β-induced arrangement of DHPRs in tetrads was not detected as no ultrastructural information was obtained.In the present study, we established zebrafish mutant relaxed as an expression system to test different β subunits for their ability to restore skeletal muscle EC coupling. Using isolated myotubes for in vitro experiments (19, 27) and complete larvae for in vivo expression studies (28-31) and freeze-fracture electron microscopy, a clear differentiation between the major functional roles of β subunits was feasible in the zebrafish system. The cloned zebrafish β_1a and a mammalian (rabbit) β_1a were shown to completely restore all parameters of EC coupling when expressed in relaxed myotubes and larvae. However, the phylogenetically closest β subunit to β_1a, the cardiac/neuronal isoform β_2a from rat, as well as the ancestral β_M isoform from the housefly (Musca domestica), could recover functional α_1S membrane insertion, but led to very restricted tetrad formation when compared with β_1a, and thus to impaired DHPR-RyR1 coupling. This impairment caused drastic changes in skeletal muscle function.The present study shows that the enhancement of functional α_1S membrane expression is a common function of all the tested β subunits, from β_1a to even the most distant β_M, whereas the effective formation of tetrads and thus proper skeletal muscle EC coupling is an exclusive function of the skeletal muscle β_1a subunit. In context with previous studies, our results suggest a model according to which β_1a acts as an allosteric modifier of α_1S conformation. Only in the presence of β_1a, the α_1S subunit is properly folded to allow RyR1 anchoring and thus skeletal muscle-type EC coupling.