Determinants in CaV1 Channels That Regulate the Ca2+ Sensitivity of Bound Calmodulin

Calmodulin binds to IQ motifs in the α₁ subunit of Ca_V1.1 and Ca_V1.2, but the affinities of calmodulin for the motif and for Ca²⁺ are higher when bound to Ca_V1.2 IQ. The Ca_V1.1 IQ and Ca_V1.2 IQ sequences differ by four amino acids. We determined the structure of calmodulin bound to Ca_V1.1 IQ and compared it with that of calmodulin bound to Ca_V1.2 IQ. Four methionines in Ca²⁺-calmodulin form a hydrophobic binding pocket for the peptide, but only one of the four nonconserved amino acids (His-1532 of Ca_V1.1 and Tyr-1675 of Ca_V1.2) contacts this calmodulin pocket. However, Tyr-1675 in Ca_V1.2 contributes only modestly to the higher affinity of this peptide for calmodulin; the other three amino acids in Ca_V1.2 contribute significantly to the difference in the Ca²⁺ affinity of the bound calmodulin despite having no direct contact with calmodulin. Those residues appear to allow an interaction with calmodulin with one lobe Ca²⁺-bound and one lobe Ca²⁺-free. Our data also provide evidence for lobe-lobe interactions in calmodulin bound to Ca_V1.2.The complexity of eukaryotic Ca²⁺ signaling arises from the ability of cells to respond differently to Ca²⁺ signals that vary in amplitude, duration, and location. A variety of mechanisms decode these signals to drive the appropriate physiological responses. The Ca²⁺ sensor for many of these physiological responses is the Ca²⁺-binding protein calmodulin (CaM).² The primary sequence of CaM is tightly conserved in all eukaryotes, yet it binds and regulates a broad set of target proteins in response to Ca²⁺ binding. CaM has two domains that bind Ca²⁺ as follows: an amino-terminal domain (N-lobe) and a carboxyl-terminal domain (C-lobe) joined via a flexible α-helix. Each lobe of CaM binds two Ca²⁺ ions, and binding within each lobe is highly cooperative. The two lobes of CaM, however, have distinct Ca²⁺ binding properties; the C-lobe has higher Ca²⁺ affinity because of a slower rate of dissociation, whereas the N-lobe has weaker Ca²⁺ affinity and faster kinetics (1). CaM can also bind to some target proteins in both the presence and absence of Ca²⁺, and the preassociation of CaM in low Ca²⁺ modulates the apparent Ca²⁺ affinity of both the amino-terminal and carboxyl-terminal lobes. Differences in the Ca²⁺ binding properties of the lobes and in the interaction sites of the amino- and carboxyl-terminal lobes enable CaM to decode local versus global Ca²⁺ signals (2).Even though CaM is highly conserved, CaM target (or recognition) sites are quite heterogeneous. The ability of CaM to bind to very different targets is at least partially due to its flexibility, which allows it to assume different conformations when bound to different targets. CaM also binds to various targets in distinct Ca²⁺ saturation states as follows: Ca²⁺-free (3), Ca²⁺ bound to only one of the two lobes, or fully Ca²⁺-bound (4–7). In addition, CaM may bind with both lobes bound to a target (5, 6) or with only a single lobe engaged (8). If a target site can bind multiple conformers of CaM, CaM may undergo several transitions that depend on Ca²⁺ concentration, thereby tuning the functional response. Identification of stable intermediate states of CaM bound to individual targets will help to elucidate the steps involved in this fine-tuned control.Both Ca_V1.1 and Ca_V1.2 belong to the L-type family of voltage-dependent Ca²⁺ channels, which bind apoCaM and Ca²⁺-CaM at carboxyl-terminal recognition sites in their α₁ subunits (9–14). Ca²⁺ binding to CaM, bound to Ca_V1.2 produces Ca²⁺-dependent facilitation (CDF) (14). Whether Ca_V1.1 undergoes CDF is not known. However, both Ca_V1.2 and Ca_V1.1 undergo Ca²⁺- and CaM-dependent inactivation (CDI) (14, 15). Ca_V1.1 CDI is slower and more sensitive to buffering by 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid than Ca_V1.2 CDI (15). Ca²⁺ buffers are thought to influence CDI and/or CDF in voltage-dependent Ca²⁺ channels by competing with CaM for Ca²⁺ (16).The conformation of the carboxyl terminus of the α₁ subunit is critical for channel function and has been proposed to regulate the gating machinery of the channel (17, 18). Several interactions of this region include intramolecular contacts with the pore inactivation machinery and intermolecular contacts with CaM kinase II and ryanodine receptors (17, 19–22). Ca²⁺ regulation of Ca_V1.2 may involve several motifs within this highly conserved region, including an EF hand motif and three contiguous CaM-binding sequences (10, 12). ApoCaM and Ca²⁺-CaM-binding sites appear to overlap at the site designated as the “IQ motif” (9, 12, 13), which are critical for channel function at the molecular and cellular level (14, 23).Differences in the rate at which 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid affects CDI of Ca_V1.1 and Ca_V1.2 could reflect differences in their interactions with CaM. In this study we describe the differences in CaM interactions with the IQ motifs of the Ca_V1.1 and the Ca_V1.2 channels in terms of crystal structure, CaM affinity, and Ca²⁺ binding to CaM. We find the structures of Ca²⁺-CaM-IQ complexes are similar except for a single amino acid change in the peptide that contributes to its affinity for CaM. We also find that the other three amino acids that differ in Ca_V1.2 and Ca_V1.1 contribute to the ability of Ca_V1.2 to bind a partially Ca²⁺-saturated form of CaM.     

Separate Elements within a Single IQ-like Motif in Adenylyl Cyclase Type 8 Impart Ca2+/Calmodulin Binding and Autoinhibition

The ubiquitous Ca²⁺-sensing protein calmodulin (CaM) fulfills its numerous signaling functions through a wide range of modular binding and activation mechanisms. By activating adenylyl cyclases (ACs) 1 and 8, Ca²⁺ acting via calmodulin impacts on the signaling of the other major cellular second messenger cAMP. In possessing two CaM-binding domains, a 1-5-8-14 motif at the N terminus and an IQ-like motif (IQlm) at the C terminus, AC8 offers particularly sophisticated regulatory possibilities. The IQlm has remained unexplored beyond the suggestion that it bound CaM, and the larger C2b region of which it is part was involved in the relief of autoinhibition of AC8. Here we attempt to distinguish the function of individual residues of the IQlm. From a complementary approach of in vitro and cell population AC activity assays, as well as CaM binding, we propose that the IQlm alone, and not the majority of the C2b, imparts CaM binding and autoinhibitory functions. Moreover, this duality of function is spatially separated and depends on amino acid side-chain character. Accordingly, residues critical for CaM binding are positively charged and clustered toward the C terminus, and those essential for the maintenance of autoinhibition are hydrophobic and more N-terminal. Secondary structure prediction of the IQlm supports this separation, with an ideally placed break in the α-helical nature of the sequence. We additionally find that the N and C termini of AC8 interact, which is an association specifically abrogated by fully Ca²⁺-bound, but not Ca²⁺-free, CaM. These data support a sophisticated activation mechanism of AC8 by CaM, in which the duality of the IQlm function is critical.The divalent calcium ion, Ca²⁺, plays a key role in modulating cellular processes as diverse as fertilization and apoptosis (1, 2). Ca²⁺ concentrations inside the cell are held ∼100 nm, despite a perpetually higher level of 1–2 mm in the extracellular medium. This steep gradient across the plasma membrane allows for a large influx when Ca²⁺ channels open, with subsequent signaling events that often rely on transduction via Ca²⁺-binding proteins (3). The archetypal Ca²⁺ sensor is calmodulin (CaM),2 a small, acidic protein so strictly conserved that all vertebrate CaM genes encode an identical 148-residue sequence (4). Multifunctional in its downstream effects, CaM can bind to at least 300 target proteins with novel partners continuing to be discovered (5). The list of effectors includes two isoforms of the adenylyl cyclase (AC) superfamily, AC1 and AC8, which comprise the Ca²⁺-stimulable subset of the nine particulate ACs (6). In intact cells, AC8 exhibits a predilection for store-operated, or capacitative, Ca²⁺ entry (CCE) (7, 8). This mode of Ca²⁺ entry is triggered by the emptying of endo/sarcoplasmic reticular stores by physiological or pharmacological stimuli (9, 10). Although the mechanism of the regulation of AC8 in nonexcitable cells by CCE (or voltage-gated Ca²⁺ entry in neurons) can be viewed to rely on facets of cellular compartmentalization (11–13), the detailed molecular mechanism whereby Ca²⁺ stimulates the enzyme is unclear. Consequently, the present investigation addresses the molecular mechanism whereby Ca²⁺, acting via calmodulin, stimulates AC8.The broad features of CaM that hold the key to its multiple regulatory strategies are understood. CaM is organized into two homologous globular domains (or lobes) united by a short linker segment (14). Both the N-terminal (N-lobe) and the C-terminal lobe (C-lobe) include two EF-hands (specialized Ca²⁺-coordinating helix-loop-helix motifs (15)), endowing CaM with four Ca²⁺-binding sites. Ca²⁺ binding to either lobe of CaM induces a structural reconfiguration determined by the two helices of each EF-hand separating from near-antiparallel to perpendicular arrays (14, 16). This exposes hydrophobic trenches in the C- and N-lobes sequentially (because the former has the highest affinity for Ca²⁺), which are notably lined with a disproportionate number of flexible methionine side chains, ready to accommodate a remarkable array of unrelated sequences. Initially, the mode of Ca²⁺-loaded CaM association with targets was considered to be uniform; the N- and C-lobes collapsed around the target peptides of e.g. smooth muscle myosin light chain kinase (17), skeletal muscle myosin light chain kinase (18), and CaMKIIα (19) with the N-lobe favoring the C-terminal target sequence and vice versa. However, CaM has since proven to be more versatile and unpredictable in how it associates with and regulates effectors, with reports of N-lobe-N-terminal/C-lobe-C-terminal interaction (20), target dimerization promoted by CaM (21), CaM binding to fatty acyl modifications (22), and other deviations from the early model.Nevertheless, three main forms of CaM regulation have been proposed as follows: relief of autoinhibition; active site remodeling; and dimerization of target domains (23). Whether the precise mechanism of CaM binding and subsequent regulation of AC8 falls into these categories of CaM regulation is not resolved. A previous study (24) established that AC8 possesses two calmodulin-binding domains (CaMBDs). CaM recognition sequences generally show little homology, although classifications based on relative positions of key hydrophobic residues have been usefully applied (4). The N-terminal CaMBD of AC8 conforms to a “1-5-8-14 motif” having large hydrophobic side chains from Trp, Val, or Ile residues at these spatially conserved sites. The C-terminal CaMBD contains an IQ-like motif (IQlm), in accordance with a signature (IVL)QXXXR(K) arrangement, to which CaM binds directly (25). By truncating one terminus or both termini, Gu and Cooper (24) asserted that the N terminus contributed little to direct CaM regulation of AC8, whereas the C terminus was critical for the maintenance of an auto-inhibited state, which was relieved upon binding of CaM. Thus, functional elements of both autoinhibition and pre-association, imparted by noncontiguous CaMBDs, have been suggested, thereby excluding AC8 from a simple model of CaM binding to an autoinhibitory domain leading to activation, as is sometimes observed (23).Recently, the proposal that the two CaMBDs play separate roles in AC8 activation was reinforced (26). This study suggested that CaM tethering by the N-terminal 1-5-8-14 motif provides the catalytically relevant C-terminal CaMBD with privileged access to CaM, thereby circumventing the need for AC8 to compete for CaM, whose free concentration in the cell is far exceeded by that of its targets (27, 28). 1–14 motifs are more generally employed in relieving autoinhibitory influences (23), so the use by AC8 of a 1-5-8-14 motif as a CaM-tethering site is unusual. The proposal that CaM pre-associates here was based on limited mutagenesis of residues within the 1-5-8-14 motif of AC8 and the use of CaM mutants whose Ca²⁺ binding capability was abolished completely or limited to discrete lobes. In contrast to the 1-5-8-14 motif at the N terminus, very little is known of the IQlm at the C terminus, in terms of the roles of the individual residues in CaM binding or autoinhibition, or even on the interplay between CaMBDs at the N and C termini of AC8.Against this background, the present study sought to assess the contribution of IQlm residues to the function of AC8, focusing on consequences of key mutations on CaM-binding efficiency, regulation by Ca²⁺/CaM, and maintenance of the autoinhibited state. Through this series of experiments, the level of coordination between the IQ-like and 1-5-8-14 motifs became evident. The provision of a pre-associated CaM molecule was found to allow for potentially deleterious mutations of the IQlm to be tolerated. Within this latter motif, Leu¹¹⁹⁶, Val¹¹⁹⁷, and Leu¹²⁰⁰ are residues essential to autoinhibition, whereas the main responsibility of binding CaM directly at the C terminus lies with Arg¹²⁰² and Arg¹²⁰⁴. In this regard, the AC8 IQlm spatially separates the two functions of CaM binding and maintenance of autoinhibition, a separation that is supported by a predicted break in the helicity of the IQlm region. Thus, a new variation is revealed in the manner by which a target exploits the CaM device into a sophisticated activation mechanism.     

Signaling Processes for Initiating Smooth Muscle Contraction upon Neural Stimulation

Relationships among biochemical signaling processes involved in Ca²⁺/calmodulin (CaM)-dependent phosphorylation of smooth muscle myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) were determined. A genetically-encoded biosensor MLCK for measuring Ca²⁺-dependent CaM binding and activation was expressed in smooth muscles of transgenic mice. We performed real-time evaluations of the relationships among [Ca²⁺]_i, MLCK activation, and contraction in urinary bladder smooth muscle strips neurally stimulated for 3 s. Latencies for the onset of [Ca²⁺]_i and kinase activation were 55 ± 8 and 65 ± 6 ms, respectively. Both increased with RLC phosphorylation at 100 ms, whereas force latency was 109 ± 3 ms. [Ca²⁺]_i, kinase activation, and RLC phosphorylation responses were maximal by 1.2 s, whereas force increased more slowly to a maximal value at 3 s. A delayed temporal response between RLC phosphorylation and force is probably due to mechanical effects associated with elastic elements in the tissue. MLCK activation partially declined at 3 s of stimulation with no change in [Ca²⁺]_i and also declined more rapidly than [Ca²⁺]_i during relaxation. The apparent desensitization of MLCK to Ca²⁺ activation appears to be due to phosphorylation in its calmodulin binding segment. Phosphorylation of two myosin light chain phosphatase regulatory proteins (MYPT1 and CPI-17) or a protein implicated in strengthening membrane adhesion complexes for force transmission (paxillin) did not change during force development. Thus, neural stimulation leads to rapid increases in [Ca²⁺]_i, MLCK activation, and RLC phosphorylation in phasic smooth muscle, showing a tightly coupled Ca²⁺ signaling complex as an elementary mechanism initiating contraction.Increases in [Ca²⁺]_i3 in smooth muscle cells lead to Ca²⁺/CaM-dependent MLCK activation and RLC phosphorylation. Phosphorylation of RLC increases actin-activated myosin MgATPase activity leading to myosin cross-bridge cycling with force development (1–3).The activation of smooth muscle contraction may be affected by multiple cellular processes. Previous investigations show that free Ca²⁺/CaM is limiting for kinase activation despite the abundance of total CaM (4–6). The extent of RLC phosphorylation is balanced by the actions of MLCK and myosin light chain phosphatase, which is composed of three distinct protein subunits (7). The myosin phosphatase targeting subunit, MYPT1, in smooth muscle binds to myosin filaments, thus targeting the 37-kDa catalytic subunit (type 1 serine/threonine phosphatase, PP1c) to phosphorylated RLC. RLC phosphorylation and muscle force may be regulated by additional signaling pathways involving phosphorylation of RLC by Ca²⁺-independent kinase(s) and inhibition of myosin light chain phosphatase, processes that increase the contraction response at fixed [Ca²⁺]_i (Ca²⁺-sensitization) (8–14). Many studies indicate that agonist-mediated Ca²⁺-sensitization most often reflects decreased myosin light chain phosphatase activity involving two major pathways including MYPT1 phosphorylation by a Rho kinase pathway and phosphorylation of CPI-17 by PKC (8, 14–16). Additionally, phosphorylation of MLCK in its calmodulin-binding sequence by a Ca²⁺/calmodulin-dependent kinase pathway has been implicated in Ca²⁺ desensitization of RLC phosphorylation (17–19). How these signaling pathways intersect the responses of the primary Ca²⁺/CaM pathway during physiological neural stimulation is not known.There is also evidence that smooth muscle contraction requires the polymerization of submembranous cytoskeletal actin filaments to strengthen membrane adhesion complexes involved in transmitting force between actin-myosin filaments and external force-transmitting structures (20–23). In tracheal smooth muscle, paxillin at membrane adhesions undergoes tyrosine phosphorylation in response to contractile stimulation by an agonist, and this phosphorylation increases concurrently with force development in response to agonist. Expression of nonphosphorylatable paxillin mutants in tracheal muscle suppresses acetylcholine-induced tyrosine phosphorylation of paxillin, tension development, and actin polymerization without affecting RLC phosphorylation (24, 25). Thus, paxillin phosphorylation may play an important role in tension development in smooth muscle independently of RLC phosphorylation and cross-bridge cycling.Specific models relating signaling mechanisms in the smooth muscle cell to contraction dynamics are limited when cells in tissues are stimulated slowly and asynchronously by agonist diffusing into the preparation. Field stimulation leading to the rapid release of neurotransmitters from nerves embedded in the tissue avoids these problems associated with agonist diffusion (26, 27). In urinary bladder smooth muscle, phasic contractions are brought about by the parasympathetic nervous system. Upon activation, parasympathetic nerve varicosities release the two neurotransmitters, acetylcholine and ATP, that bind to muscarinic and purinergic receptors, respectively. They cause smooth muscle contraction by inducing Ca²⁺ transients as elementary signals in the process of nerve-smooth muscle communication (28–30). We recently reported the development of a genetically encoded, CaM-sensor for activation of MLCK. The CaM-sensor MLCK contains short smooth muscle MLCK fused to two fluorophores, enhanced cyan fluorescent protein and enhanced yellow fluorescent protein, linked by the MLCK calmodulin binding sequence (6, 14, 31). Upon dimerization, there is significant FRET from the donor enhanced cyan fluorescent protein to the acceptor enhanced yellow fluorescent protein. Ca²⁺/CaM binding dissociates the dimer resulting in a decrease in FRET intensity coincident with activation of kinase activity (31). Thus, CaM-sensor MLCK is capable of directly monitoring Ca²⁺/CaM binding and activation of the kinase in smooth muscle tissues where it is expressed specifically in smooth muscle cells of transgenic mice. We therefore combined neural stimulation with real-time measurements of [Ca²⁺]_i, MLCK activation, and force development in smooth muscle tissue from these mice. Additionally, RLC phosphorylation was measured precisely at specific times following neural stimulation in tissues frozen by a rapid-release electronic freezing device (26, 27). Results from these studies reveal that physiological stimulation of smooth muscle cells by neurotransmitter release leads to rapid increases in [Ca²⁺]_i, MLCK activation, and RLC phosphorylation at similar rates without the apparent activities of Ca²⁺-independent kinases, inhibition of myosin light chain phosphatase, or paxillin phosphorylation. Thus, the elemental processes for phasic smooth muscle contraction are represented by this tightly coupled Ca²⁺ signaling complex.     

Time-dependent Autoinactivation of Phospho-Thr286-��Ca2+/Calmodulin-dependent Protein Kinase II

Ca²⁺/calmodulin-dependent protein kinase II (αCaMKII) is thought to exert its role in memory formation by autonomous Ca²⁺-independent persistent activity conferred by Thr²⁸⁶ autophosphorylation, allowing the enzyme to remain active even when intracellular [Ca²⁺] has returned to resting levels. Ca²⁺ sequestration-induced inhibition, caused by a burst of Thr^305/306 autophosphorylation via calmodulin (CaM) dissociation from the Thr^305/306 sites, is in conflict with this view. The processes of CaM binding, autophosphorylation, and inactivation are dissected to resolve this conflict. Upon Ca²⁺ withdrawal, CaM sequential domain dissociation is observed, starting with the rapid release of the first (presumed N-terminal) CaM lobe, thought to be bound at the Thr^305/306 sites. The time courses of Thr^305/306 autophosphorylation and inactivation, however, correlate with the slow dissociation of the second (presumed C-terminal) CaM lobe. Exposure of the Thr^305/306 sites is thus not sufficient for their autophosphorylation. Moreover, Thr^305/306 autophosphorylation and autoinactivation are shown to occur in the continuous presence of Ca²⁺ and bound Ca²⁺/CaM by time courses similar to those seen following Ca²⁺ sequestration. Our investigation of the activity and mechanisms of phospho-Thr₂₈₆-αCaMKII thus shows time-dependent autoinactivation, irrespective of the continued presence of Ca²⁺ and CaM, allowing a very short, if any, time window for Ca²⁺/CaM-free phospho-Thr²⁸⁶-αCaMKII activity. Physiologically, the time-dependent autoinactivation mechanisms of phospho-Thr²⁸⁶-αCaMKII (t½ of ∼50 s at 37 °C) suggest a transient kinase activity of ∼1 min duration in the induction of long term potentiation and thus memory formation.Ca²⁺/calmodulin-dependent protein kinase II (αCaMKII)² is essential in hippocampal learning and N-methyl-d-aspartate receptor-dependent synaptic plasticity, causing long term potentiation (1, 2). The exact mechanisms of αCaMKII in memory functions have not yet been identified.αCaMKII is a broad specificity Ser/Thr protein kinase, which catalyzes the phosphorylation of over 100 protein and peptide substrates in vitro (3). Uniquely, the CaMKII family possesses two distinct kinase mechanisms. The first mechanism is a “canonical” intrasubunit phosphorylation, commonly found in monomeric kinases, in which the phosphorylatable residue of the substrate bound to the helical subdomain of the catalytic domain at the active site is lined up with the terminal phosphate of ATP (4). Although there is a large number of potential “canonical” substrates for αCaMKII at the synapse (5), so far AMPA receptors have been shown to be possible physiological substrates of αCaMKII (6). For the purpose of this study, syntide 2, a commonly used peptide substrate derived from phosphorylation site 2 of glycogen synthase (7), was chosen.The second mechanism, intersubunit autophosphorylation, takes advantage of the oligomeric organization of CaMKII (8). The most important autophosphorylation site in the α isoform is Thr²⁸⁶, which resides in the vicinity of the autoinhibitory domain (9). Peptide substrates with homologous sequences to this region have been reported to be phosphorylated by αCaMKII. This, however, occurs with a low V_max, and these substrates show properties of a non-competitive inhibitor with respect to phosphorylation of “canonical” substrates (10) and of Thr²⁸⁶ autophosphorylation itself (11). Examples of such substrates include autocamtide, a peptide substrate derived from the autoinhibitory region (12) and the NR2B subunit of the N-methyl-d-aspartate receptor, which has been identified as a potential physiological target of phospho-Thr²⁸⁶-αCaMKII at the postsynaptic membrane (13). The possible physiological significance of NR2B phosphorylation is not yet known. There is evidence to suggest that Thr²⁸⁶ autophosphorylation is required to achieve full activity of the enzyme, since the unphosphorylatable T286A mutant enzyme has much diminished activity compared with wild type enzyme (14, 15).Thr²⁸⁶ autophosphorylation causes CaM “trapping,” a >10⁴-fold increase in the affinity of αCaMKII for Ca²⁺/CaM (16–18). At the same time, Thr²⁸⁶ autophosphorylation is also attributed to confer Ca²⁺- and CaM-independent persistent “autonomous” kinase activity to αCaMKII. However, due to the extremely high affinity of phospho-Thr₂₈₆-αCaMKII for Ca²⁺/CaM, [Ca²⁺] of <10 nm is required to achieve full dissociation of Ca²⁺/CaM, since CaM trapping occurs by virtue of Ca²⁺ trapping (19). Partial activity measured upon partial Ca²⁺ withdrawal therefore may not always reflect Ca²⁺/CaM-free enzyme (9). Furthermore, the physiological resting [Ca²⁺] range is 50–100 nm; therefore, phospho-Thr²⁸⁶-αCaMKII is likely always to have residual Ca²⁺/CaM bound. This may be partially Ca²⁺-saturated CaM (19).Persistent autonomous activity conferred by Thr²⁸⁶ autophosphorylation is thought to enable αCaMKII to function as a memory molecule (20, 21). In contrast, however, following the development of chemical long term potentiation, rapid inactivation has also been reported (22). The extent of an autonomous activity is further obscured by the finding that Ca²⁺ sequestration induces a burst of autophosphorylation at residues Thr^305/306, followed by a loss of activity (23). Moreover, when examined across a broad range of [Ca²⁺], the Ca²⁺/CaM dependence of phospho-Thr²⁸⁶-αCaMKII activity is apparent (19). It is thus vital to establish the mechanisms of activation and inactivation of αCaMKII at the molecular level in order to understand how it may function physiologically in learning and memory. To this end, it is necessary to dissect the mechanisms of Ca²⁺/CaM dissociation, Thr^305/306 autophosphorylation, and inactivation of phospho-Thr²⁸⁶-αCaMKII and to establish the time window for autonomous Ca²⁺/CaM-independent activity.     

Interactions between Calmodulin, Adenosine A2A, and Dopamine D2 Receptors

The Ca²⁺-binding protein calmodulin (CaM) has been shown to bind directly to cytoplasmic domains of some G protein-coupled receptors, including the dopamine D₂ receptor. CaM binds to the N-terminal portion of the long third intracellular loop of the D₂ receptor, within an Arg-rich epitope that is also involved in the binding to G_i/o proteins and to the adenosine A_2A receptor, with the formation of A_2A-D₂ receptor heteromers. In the present work, by using proteomics and bioluminescence resonance energy transfer (BRET) techniques, we provide evidence for the binding of CaM to the A_2A receptor. By using BRET and sequential resonance energy transfer techniques, evidence was obtained for CaM-A_2A-D₂ receptor oligomerization. BRET competition experiments indicated that, in the A_2A-D₂ receptor heteromer, CaM binds preferentially to a proximal C terminus epitope of the A_2A receptor. Furthermore, Ca²⁺ was found to induce conformational changes in the CaM-A_2A-D₂ receptor oligomer and to selectively modulate A_2A and D₂ receptor-mediated MAPK signaling in the A_2A-D₂ receptor heteromer. These results may have implications for basal ganglia disorders, since A_2A-D₂ receptor heteromers are being considered as a target for anti-parkinsonian agents.G-protein-coupled receptors are able to form homo- and hetero-oligomers with unique biochemical and functional characteristics (1–7), and they are easily detected in vitro by using biophysical techniques (8–10). Heteromers of adenosine A_2A and dopamine D₂ receptors were one of the first G-protein-coupled receptor heteromers to be described (11). A close physical interaction between both receptors was shown using co-immunoprecipitation and co-localization assays (11) and fluorescence and bioluminescence resonance energy transfer (FRET² or BRET) techniques (12–14). At the biochemical level, two types of antagonistic A_2A-D₂ receptor interactions have been discovered that may explain the A_2A-D₂ receptor interactions described both at the neuronal and behavioral level (11, 15–18). First, by means of an allosteric interaction in the receptor heteromer, stimulation of A_2A receptor decreases the affinity of D₂ receptor for their agonists (12). Second, the stimulation of the G_i/o-protein-coupled D₂ receptor inhibits the cAMP accumulation induced by the stimulation of the G_s/olf-protein-coupled A_2A receptor (11, 17, 18). In view of the well known role of dopamine in Parkinson disease, schizophrenia, and drug addiction, it has been suggested that the A_2A-D₂ receptor interactions in the central nervous system may provide new therapeutic approaches to combat these disorders (16, 19).An epitope-epitope electrostatic interaction between an Arg-rich epitope of the N terminus of the third intracellular loop (3IL) of the D₂ receptor and an epitope containing a phosphorylated Ser localized in the distal part of the C terminus of the A_2A receptor is involved in A_2A-D₂ receptor heteromer interface (14, 20, 21). The same Arg-rich epitope of the D₂ receptor is able to interact with CaM (22–25). In the absence of phosphorylated residues, adjacent aspartates or glutamates, which are abundant in CaM, may also form non-covalent complexes with Arg-rich epitopes (26). Therefore, CaM can potentially convey a Ca²⁺ signal to the D₂ receptor through direct binding to the 3IL of the D₂ receptor (22). Mass spectrometry data have shown that bovine CaM can form multiple non-covalent complexes with an Arg-rich peptide corresponding to the N-terminal region of the 3IL of the D₂ receptor (VLRRRRKRVN) (24) as well as a peptide from the proximal C terminus of the A_2A receptor (24). This epitope, whose sequence is ²⁹¹RIREFRQTFR³⁰⁰ in the human A_2A receptor, also contains several Arg residues. Since the suspected interaction between the A_2A receptor and CaM was awaiting confirmation by assays using complete proteins, the present study was undertaken to demonstrate the existence of interactions between the A_2A receptor and CaM both in a recombinant protein expression cell system and in the brain. A proteomics approach was used for the discovery of protein-protein interactions between the A_2A receptor and CaM in rat brain, whereas BRET in transfected cells demonstrated a direct interaction between CaM and this receptor. Furthermore, by using BRET and sequential resonance energy transfer (SRET) techniques and analyzing MAPK signaling in transfected cells, evidence was obtained for CaM-A_2A-D₂ receptor oligomerization and a selective Ca²⁺-mediated modulation of A_2A and D₂ receptor function in the A_2A-D₂ receptor heteromer.     

Role of Partitioning-defective 1/Microtubule Affinity-regulating Kinases in the Morphogenetic Activity of Helicobacter pylori CagA

Helicobacter pylori CagA plays a key role in gastric carcinogenesis. Upon delivery into gastric epithelial cells, CagA binds and deregulates SHP-2 phosphatase, a bona fide oncoprotein, thereby causing sustained ERK activation and impaired focal adhesions. CagA also binds and inhibits PAR1b/MARK2, one of the four members of the PAR1 family of kinases, to elicit epithelial polarity defect. In nonpolarized gastric epithelial cells, CagA induces the hummingbird phenotype, an extremely elongated cell shape characterized by a rear retraction defect. This morphological change is dependent on CagA-deregulated SHP-2 and is thus thought to reflect the oncogenic potential of CagA. In this study, we investigated the role of the PAR1 family of kinases in the hummingbird phenotype. We found that CagA binds not only PAR1b but also other PAR1 isoforms, with order of strength as follows: PAR1b > PAR1d ≥ PAR1a > PAR1c. Binding of CagA with PAR1 isoforms inhibits the kinase activity. This abolishes the ability of PAR1 to destabilize microtubules and thereby promotes disassembly of focal adhesions, which contributes to the hummingbird phenotype. Consistently, PAR1 knockdown potentiates induction of the hummingbird phenotype by CagA. The morphogenetic activity of CagA was also found to be augmented through inhibition of non-muscle myosin II. Because myosin II is functionally associated with PAR1, perturbation of PAR1-regulated myosin II by CagA may underlie the defect of rear retraction in the hummingbird phenotype. Our findings reveal that CagA systemically inhibits PAR1 family kinases and indicate that malfunctioning of microtubules and myosin II by CagA-mediated PAR1 inhibition cooperates with deregulated SHP-2 in the morphogenetic activity of CagA.Infection with Helicobacter pylori strains bearing cagA (cytotoxin-associated gene A)-positive strains is the strongest risk factor for the development of gastric carcinoma, the second leading cause of cancer-related death worldwide (1–3). The cagA gene is located within a 40-kb DNA fragment, termed the cag pathogenicity island, which is specifically present in the genome of cagA-positive H. pylori strains (4–6). In addition to cagA, there are ∼30 genes in the cag pathogenicity island, many of which encode a bacterial type IV secretion system that delivers the cagA-encoded CagA protein into gastric epithelial cells (7–10). Upon delivery into gastric epithelial cells, CagA is localized to the plasma membrane, where it undergoes tyrosine phosphorylation at the C-terminal Glu-Pro-Ile-Tyr-Ala motifs by Src family kinases or c-Abl kinase (11–14). The C-terminal Glu-Pro-Ile-Tyr-Ala-containing region of CagA is noted for the structural diversity among distinct H. pylori isolates. Oncogenic potential of CagA has recently been confirmed by a study showing that systemic expression of CagA in mice induces gastrointestinal and hematological malignancies (15).When expressed in gastric epithelial cells, CagA induces morphological transformation termed the hummingbird phenotype, which is characterized by the development of one or two long and thin protrusions resembling the beak of the hummingbird. It has been thought that the hummingbird phenotype is related to the oncogenic action of CagA (7, 16–19). Pathophysiological relevance for the hummingbird phenotype in gastric carcinogenesis has recently been provided by the observation that infection with H. pylori carrying CagA with greater ability to induce the hummingbird phenotype is more closely associated with gastric carcinoma (20–23). Elevated motility of hummingbird cells (cells showing the hummingbird phenotype) may also contribute to invasion and metastasis of gastric carcinoma.In host cells, CagA interacts with the SHP-2 phosphatase, C-terminal Src kinase, and Crk adaptor in a tyrosine phosphorylation-dependent manner (16, 24, 25) and also associates with Grb2 adaptor and c-Met in a phosphorylation-independent manner (26, 27). Among these CagA targets, much attention has been focused on SHP-2 because the phosphatase has been recognized as a bona fide oncoprotein, gain-of-function mutations of which are found in various human malignancies (17, 18, 28). Stable interaction of CagA with SHP-2 requires CagA dimerization, which is mediated by a 16-amino acid CagA-multimerization (CM)² sequence present in the C-terminal region of CagA (29). Upon complex formation, CagA aberrantly activates SHP-2 and thereby elicits sustained ERK MAP kinase activation that promotes mitogenesis (30). Also, CagA-activated SHP-2 dephosphorylates and inhibits focal adhesion kinase (FAK), causing impaired focal adhesions. It has been shown previously that both aberrant ERK activation and FAK inhibition by CagA-deregulated SHP-2 are involved in induction of the hummingbird phenotype (31).Partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK) is an evolutionally conserved serine/threonine kinase originally isolated in C. elegans (32–34). Mammalian cells possess four structurally related PAR1 isoforms, PAR1a/MARK3, PAR1b/MARK2, PAR1c/MARK1, and PAR1d/MARK4 (35–37). Among these, PAR1a, PAR1b, and PAR1c are expressed in a variety of cells, whereas PAR1d is predominantly expressed in neural cells (35, 37). These PAR1 isoforms phosphorylate microtubule-associated proteins (MAPs) and thereby destabilize microtubules (35, 38), allowing asymmetric distribution of molecules that are involved in the establishment and maintenance of cell polarity.In polarized epithelial cells, CagA disrupts the tight junctions and causes loss of apical-basolateral polarity (39, 40). This CagA activity involves the interaction of CagA with PAR1b/MARK2 (19, 41). CagA directly binds to the kinase domain of PAR1b in a tyrosine phosphorylation-independent manner and inhibits the kinase activity. Notably, CagA binds to PAR1b via the CM sequence (19). Because PAR1b is present as a dimer in cells (42), CagA may passively homodimerize upon complex formation with the PAR1 dimer via the CM sequence, and this PAR1-directed CagA dimer would form a stable complex with SHP-2 through its two SH2 domains.Because of the critical role of CagA in gastric carcinogenesis (7, 16–19), it is important to elucidate the molecular basis underlying the morphogenetic activity of CagA. In this study, we investigated the role of PAR1 isoforms in induction of the hummingbird phenotype by CagA, and we obtained evidence that CagA-mediated inhibition of PAR1 kinases contributes to the development of the morphological change by perturbing microtubules and non-muscle myosin II.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Inflammasome-dependent Caspase-1 Activation in Cervical Epithelial Cells Stimulates Growth of the Intracellular Pathogen Chlamydia trachomatis

Inflammasomes have been extensively characterized in monocytes and macrophages, but not in epithelial cells, which are the preferred host cells for many pathogens. Here we show that cervical epithelial cells express a functional inflammasome. Infection of the cells by Chlamydia trachomatis leads to activation of caspase-1, through a process requiring the NOD-like receptor family member NLRP3 and the inflammasome adaptor protein ASC. Secretion of newly synthesized virulence proteins from the chlamydial vacuole through a type III secretion apparatus results in efflux of K⁺ through glibenclamide-sensitive K⁺ channels, which in turn stimulates production of reactive oxygen species. Elevated levels of reactive oxygen species are responsible for NLRP3-dependent caspase-1 activation in the infected cells. In monocytes and macrophages, caspase-1 is involved in processing and secretion of pro-inflammatory cytokines such as interleukin-1β. However, in epithelial cells, which are not known to secrete large quantities of interleukin-1β, caspase-1 has been shown previously to enhance lipid metabolism. Here we show that, in cervical epithelial cells, caspase-1 activation is required for optimal growth of the intracellular chlamydiae.Chlamydia trachomatis is the most common cause of bacterial sexually transmitted disease in the United States, and it is the leading cause of preventable blindness in the world (1–5). Untreated, C. trachomatis infection in women can cause pelvic inflammatory disease, which can lead to infertility and ectopic pregnancy because of scarring of the ovaries and the Fallopian tubes (6). Infection by the lymphogranuloma venereum (LGV)² strain of C. trachomatis, which has become more common in North America and Europe (7, 8), is characterized by swelling and inflammation of the lymph nodes in the groin (9).Chlamydiae are intracellular pathogens that preferentially infect epithelial mucosa and have a biphasic infection cycle (10). A metabolically inactive form, the elementary body, infects the epithelial host cells through entry vesicles that avoid fusion with host cell lysosomes and develop into a membrane-bound inclusion (11–13). Despite their intravacuolar localization, chlamydiae are still able to acquire nutrients from the host cell and interact with host-cell signaling pathways (13–23). Within a few hours, the elementary bodies differentiate into larger, metabolically active reticulate bodies, which proliferate but are noninfectious. Depending on the strain of C. trachomatis, the reticulate bodies transform back into elementary bodies after 1–3 days and are released into the extracellular medium to infect other cells (11, 24, 25). Chlamydial species possess a type III secretion (T3S) system that secretes bacterial virulence factors into host cell cytosol and may control interactions between the inclusion and host-cell compartments (26).Long before the adaptive immune response is activated, infected epithelial cells produce proinflammatory cytokines and chemokines, including interleukin (IL)-6, IL-8, and granulocyte-macrophage colony-stimulating factor (27), which recruit neutrophils to the site of infection and activate other immune effector cells. However, in many cases the immune system fails to clear the infection, and the chronic release of cytokines becomes a major contributor to the scarring and damage associated with the infection (28–30).The innate immune response during C. trachomatis infection is initiated by chlamydial pathogen-associated molecular patterns, including lipopolysaccharides, which bind to pattern recognition receptors such as Toll-like receptors and cytosolic NOD-like receptors (NLRs), ultimately promoting pro-inflammatory cytokine gene expression and secretion of the cytokine proteins (31–37). However, secretion of the key pro-inflammatory cytokine IL-1β is tightly regulated (38). First, pro-IL-1β is produced following activation of pattern recognition receptor, and the precursor is then cleaved into the mature form by the pro-inflammatory cysteine protease, caspase-1 (also known as interleukin-1 converting enzyme or ICE). The mechanism by which caspase-1 is activated in response to infection or tissue damage was found to be modulated by a macromolecular protein complex termed the “inflammasome,” which consists of an NLR family member, an adaptor protein (apoptosis-associated speck-like protein containing a caspase activation recruitment domain or ASC), and an inactive caspase-1 precursor (pro-caspase-1) (39, 40). Previous studies demonstrated that IL-1β is produced in response to chlamydial infection in dendritic cells, macrophages, and monocytes (41–44). Moreover, C. trachomatis or Chlamydia caviae infection activates caspase-1 in epithelial cells or monocytes (43, 45, 46). However, whether caspase-1 activation during chlamydial infection requires the formation of an inflammasome remains unclear.Previous studies have shown that different pathogens can cause inflammasome-mediated caspase-1 activation in macrophages and monocytes (47). However, epithelial cells lining mucosal surfaces are not only the preferred target for chlamydial infection and other intracellular pathogens but also play an important role in early host immune response to infection by secreting proinflammatory cytokines and chemokines (27). Although epithelial cells are not known to secrete large amounts of IL-1β, inflammasome-dependent caspase-1 activation in epithelial cells is known to contribute to lipid metabolism and membrane regeneration in epithelial cells damaged by the membrane-disrupting toxin, aerolysin (48). As lipids are sorted from the Golgi apparatus to the chlamydial inclusion (13, 15, 49), we therefore investigated whether C. trachomatis induces caspase-1 activation in epithelial cells via the assembly of an inflammasome. We demonstrated that C. trachomatis-induced caspase-1 activation is mediated by an inflammasome containing the NLR member, NLRP3. Several studies have demonstrated the involvement of T3S apparatus in inflammasome-mediated caspase-1 activation by different pathogens in macrophages and monocytes (50–56). Therefore, we further investigated the mechanism by which C. trachomatis triggers the formation of the NLRP3 inflammasome. Our results showed that metabolically active chlamydiae, relying on their T3S apparatus, cause K⁺ efflux, which in turn leads to formation of reactive oxygen species (ROS) and ultimately NLRP3-dependent caspase-1 activation. Epithelial cells do not typically secrete large amounts of IL-1β; instead, caspase-1 activation in cervical epithelial cells contributes to development of the chlamydial inclusion.     

Defining the Structural Basis of Human Plasminogen Binding by Streptococcal Surface Enolase

The flesh-eating bacterium group A Streptococcus (GAS) binds and activates human plasminogen, promoting invasive disease. Streptococcal surface enolase (SEN), a glycolytic pathway enzyme, is an identified plasminogen receptor of GAS. Here we used mass spectrometry (MS) to confirm that GAS SEN is octameric, thereby validating in silico modeling based on the crystal structure of Streptococcus pneumoniae α-enolase. Site-directed mutagenesis of surface-located lysine residues (SEN^{K252 + 255A}, SEN^K304A, SEN^K334A, SEN^K344E, SEN^K435L, and SEN^Δ434–435) was used to examine their roles in maintaining structural integrity, enzymatic function, and plasminogen binding. Structural integrity of the GAS SEN octamer was retained for all mutants except SEN^K344E, as determined by circular dichroism spectroscopy and MS. However, ion mobility MS revealed distinct differences in the stability of several mutant octamers in comparison with wild type. Enzymatic analysis indicated that SEN^K344E had lost α-enolase activity, which was also reduced in SEN^K334A and SEN^Δ434–435. Surface plasmon resonance demonstrated that the capacity to bind human plasminogen was abolished in SEN^{K252 + 255A}, SEN^K435L, and SEN^Δ434–435. The lysine residues at positions 252, 255, 434, and 435 therefore play a concerted role in plasminogen acquisition. This study demonstrates the ability of combining in silico structural modeling with ion mobility-MS validation for undertaking functional studies on complex protein structures.Streptococcus pyogenes (group A Streptococcus, GAS)⁸ is a common bacterial pathogen, causing over 700 million human disease episodes each year (1). These range from serious life-threatening invasive diseases including necrotizing fasciitis and streptococcal toxic shock-like syndrome to non-invasive infections like pharyngitis and pyoderma. Invasive disease, in combination with postinfection immune sequelae including rheumatic heart disease and acute poststreptococcal glomerulonephritis, account for over half a million deaths each year (1). Although a resurgence of GAS invasive infections has occurred in western countries since the mid-1980s, disease burden is much greater in developing countries and indigenous populations of developed nations, where GAS infections are endemic (2–4).GAS is able to bind human plasminogen and activate the captured zymogen to the serine protease plasmin (5–17). The capacity of GAS to do this plays a critical role in virulence and invasive disease initiation (3, 17–19). The plasminogen activation system in humans is an important and highly regulated process that is responsible for breakdown of extracellular matrix components, dissolution of blood clots, and cell migration (20, 21). Plasminogen is a 92-kDa zymogen that circulates in human plasma at a concentration of 2 μm (22). It consists of a binding region of five homologous triple loop kringle domains and an N-terminal serine protease domain that flank the Arg⁵⁶¹–Val⁵⁶² site (23), where it is cleaved by tissue plasminogen activator and urokinase plasminogen activator to yield the active protease plasmin (20, 23). GAS also has the ability to activate human plasminogen by secreting the virulence determinant streptokinase. Streptokinase forms stable complexes with plasminogen or plasmin, both of which exhibit plasmin activity (20, 24). Activation of plasminogen by the plasmin(ogen)-streptokinase complex circumvents regulation by the host plasminogen activation inhibitors, α₂-antiplasmin and α₂-macroglobulin (11, 20). GAS can bind the plasmin(ogen)-streptokinase complex and/or plasmin(ogen) directly via plasmin(ogen) receptors at the bacterial cell surface (6). These receptors include the plasminogen-binding group A streptococcal M-like protein (PAM) (25), the PAM-related protein (19), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; also known as streptococcal plasmin receptor, Plr, or streptococcal surface dehydrogenase) (9, 26), and streptococcal surface enolase (SEN or α-enolase) (27). Interactions with these GAS receptors occurs via lysine-binding sites within the kringle domains of plasminogen (6).In addition to its ability to bind human plasminogen, SEN is primarily the glycolytic enzyme that converts 2-phosphoglycerate to phosphoenolpyruvate (27–29). SEN is abundantly expressed in the cytosol of most bacterial species but has also been identified as a surface-located protein in GAS and other bacteria including pneumococci, despite lacking classical cell surface protein motifs such as a signal sequence, membrane-spanning domain, or cell-wall anchor motif (27, 28, 30, 31). The interaction between SEN and plasminogen is reported to be facilitated by the two C-terminal lysine residues at positions 434 and 435 (27, 32). In contrast, an internal binding motif containing lysines at positions 252 and 255 in the closely related α-enolase of Streptococcus pneumoniae has been shown to play a pivotal role in the acquisition of plasminogen in this bacterial species (33). The octameric pneumococcal α-enolase structure consists of a tetramer of dimers. Hence, potential binding sites could be buried in the interface between subunits. In fact, the crystal structure of S. pneumoniae α-enolase revealed that the two C-terminal lysine residues are significantly less exposed than the internal plasminogen-binding motif (34).In this study, we constructed an in silico model of GAS SEN, based on the pneumococcal octameric α-enolase crystal structure, and validated this model using ion mobility (IM) mass spectrometry (MS). Site-directed mutagenesis followed by structural and functional analyses revealed that Lys³⁴⁴ plays a crucial role in structural integrity and enzymatic function. Furthermore, we demonstrate that the plasminogen-binding motif residues Lys²⁵² and Lys²⁵⁵ and the C-terminal Lys⁴³⁴ and Lys⁴³⁵ residues are located adjacently in the GAS SEN structure and play a concerted role in the binding of human plasminogen.     

A Ca2+ Release-activated Ca2+ (CRAC) Modulatory Domain (CMD) within STIM1 Mediates Fast Ca2+-dependent Inactivation of ORAI1 Channels

STIM1 and ORAI1, the two limiting components in the Ca²⁺ release-activated Ca²⁺ (CRAC) signaling cascade, have been reported to interact upon store depletion, culminating in CRAC current activation. We have recently identified a modulatory domain between amino acids 474 and 485 in the cytosolic part of STIM1 that comprises 7 negatively charged residues. A STIM1 C-terminal fragment lacking this domain exhibits enhanced interaction with ORAI1 and 2–3-fold higher ORAI1/CRAC current densities. Here we focused on the role of this CRAC modulatory domain (CMD) in the fast inactivation of ORAI1/CRAC channels, utilizing the whole-cell patch clamp technique. STIM1 mutants either with C-terminal deletions including CMD or with 7 alanines replacing the negative amino acids within CMD gave rise to ORAI1 currents that displayed significantly reduced or even abolished inactivation when compared with STIM1 mutants with preserved CMD. Consistent results were obtained with cytosolic C-terminal fragments of STIM1, both in ORAI1-expressing HEK 293 cells and in RBL-2H3 mast cells containing endogenous CRAC channels. Inactivation of the latter, however, was much more pronounced than that of ORAI1. The extent of inactivation of ORAI3 channels, which is also considerably more prominent than that of ORAI1, was also substantially reduced by co-expression of STIM1 constructs missing CMD. Regarding the dependence of inactivation on Ca²⁺, a decrease in intracellular Ca²⁺ chelator concentrations promoted ORAI1 current fast inactivation, whereas Ba²⁺ substitution for extracellular Ca²⁺ completely abrogated it. In summary, CMD within the STIM1 cytosolic part provides a negative feedback signal to Ca²⁺ entry by triggering fast Ca²⁺-dependent inactivation of ORAI/CRAC channels.The Ca²⁺ release-activated Ca²⁺ (CRAC)⁵ channel is one of the best characterized store-operated entry pathways (1–7). Substantial efforts have led to identification of two key components of the CRAC channel machinery: the stromal interaction molecule 1 (STIM1), which is located in the endoplasmic reticulum and acts as a Ca²⁺ sensor (8–10), and ORAI1/CRACM1, the pore-forming subunit of the CRAC channel (11–13). Besides ORAI1, two further homologues named ORAI2 and ORAI3 belong to the ORAI channel family (12, 14).STIM1 senses endoplasmic reticulum store depletion primarily by its luminal EF-hand in its N terminus (8, 15), redistributes close to the plasma membrane, where it forms puncta-like structures, and co-clusters with ORAI1, leading to inward Ca²⁺ currents (12, 16–19). The STIM1 C terminus, located in the cytosol, contains two coiled-coil regions overlapping with an ezrin-radixin-moesin (ERM)-like domain followed by a serine/proline- and a lysine-rich region (2, 8, 20–22). Three recent studies have described the essential ORAI-activating region within the ERM domain, termed SOAR (Stim ORAI-activating region) (23), OASF (ORAI-activating small fragment) (24), and CAD (CRAC-activating domain) (25), including the second coiled coil domain and the following ∼55 amino acids. We and others have provided evidence that store depletion leads to a dynamic coupling of STIM1 to ORAI1 (26–28) that is mediated by a direct interaction of the STIM1 C terminus with ORAI1 C terminus probably involving the putative coiled-coil domain in the latter (27).Furthermore, different groups have proven that the C terminus of STIM1 is sufficient to activate CRAC as well as ORAI1 channels independent of store depletion (22–25, 27, 29). We have identified that OASF-(233–474) or shorter fragments exhibit further enhanced coupling to ORAI1 resulting in 3-fold increased constitutive Ca²⁺ currents. A STIM1 fragment containing an additional cluster of anionic amino acids C-terminal to position 474 displays weaker interaction with ORAI1 as well as reduced Ca²⁺ current comparable with that mediated by wild-type STIM1 C terminus. Hence, we have suggested that these 11 amino acids (474–485) act in a modulatory manner onto ORAI1; however, their detailed mechanistic impact within the STIM1/ORAI1 signaling machinery has remained so far unclear.In this study, we focused on the impact of this negative cluster on fast inactivation of STIM1-mediated ORAI Ca²⁺ currents. Lis et al. (30) have shown that all three ORAI homologues display distinct inactivation profiles, where ORAI2 and ORAI3 show a much more pronounced fast inactivation than ORAI1. Moreover, it has been reported (31) that different expression levels of STIM1 to ORAI1 affect the properties of CRAC current inactivation. Yamashita et al. (32) have demonstrated a linkage between the selectivity filter of ORAI1 and its Ca²⁺-dependent fast inactivation. Here we provide evidence that a cluster of acidic residues within the C terminus of STIM1 is involved in the fast inactivation of ORAI1 and further promotes that of ORAI3 and native CRAC currents.