GPI-anchored receptor clusters transiently recruit Lyn and G alpha for temporary cluster immobilization and Lyn activation: single-molecule tracking study 1

The signaling mechanisms for glycosylphosphatidylinositol-anchored receptors (GPI-ARs) have been investigated by tracking single molecules in living cells. Upon the engagement or colloidal gold-induced cross-linking of CD59 (and other GPI-ARs) at physiological levels, CD59 clusters containing three to nine CD59 molecules were formed, and single molecules of Galphai2 or Lyn (GFP conjugates) exhibited the frequent but transient (133 and 200 ms, respectively) recruitment to CD59 clusters, via both protein-protein and lipid-lipid (raft) interactions. Each CD59 cluster undergoes alternating periods of actin-dependent temporary immobilization (0.57-s lifetime; stimulation-induced temporary arrest of lateral diffusion [STALL], inducing IP(3) production) and slow diffusion (1.2 s). STALL of a CD59 cluster was induced right after the recruitment of Galphai2. Because both Galphai2 and Lyn are required for the STALL, and because Lyn is constitutively recruited to CD59 clusters, the STALL of CD59 clusters is likely induced by the Galphai2 binding to, and its subsequent activation of, Lyn within the same CD59 cluster.     

Galphas family G proteins activate IP(3)-Ca(2+) signaling via gbetagamma and transduce ventralizing signals in Xenopus

During early embryonic development, IP(3)-Ca(2+) signaling transduces ventral signaling at the time of dorsoventral axis formation. To identify molecules functioning upstream in this signal pathway, we examined effects of a panel of inhibitory antibodies against Galphaq/11, Galphas/olf, or Galphai/o/t/z. While all these antibodies showed direct inhibition of their targets, their effects on redirection of the ventral mesoderm to a dorsal fate varied. Anti-Galphas/olf antibody showed strong induction of dorsal fate, anti-Galphai/o/t/z antibody did so weakly, and anti-Galphaq/11 antibody was without effect. Injection of betaARK, a Gbetagamma inhibitor, mimicked the dorsalizing effect of anti-Galphas/olf antibody, whereas injection of adenylyl cyclase inhibitors at a concentration which inhibited Galphas-coupled cAMP increase did not do so. The activation of Galphas-coupled receptor gave rise to Ca(2+) transients. All these results suggest that activation of the Galphas-coupled receptor relays dorsoventral signal to Gbetagamma, which then stimulates PLCbeta and then the IP(3)-Ca(2+) system. This signaling pathway may play a crucial role in transducing ventral signals.     

Calcium mobilization evoked by hepatocellular swelling is linked to activation of phospholipase Cgamma

Recovery from swelling of hepatocytes and selected other epithelia is triggered by intracellular Ca(2+) release from the endoplasmic reticulum, which leads to fluid and electrolyte efflux through volume-sensitive K(+) and Cl(-) channels. The aim of this study was to determine the mechanisms responsible for swelling-mediated hepatocellular Ca(2+) mobilization. Swelling of HTC rat hepatoma cells, evoked by exposure to hypotonic medium, elicited transient increases in intracellular levels of inositol 1,4,5-trisphosphate (IP(3)) and cytosolic [Ca(2+)]. The latter was attenuated by inhibition of phospholipase C (PLC) with and by IP(3) receptor blockade with 2-aminoethoxydiphenyl borate, but it was unaffected by ryanodine, an inhibitor of intracellular Ca(2+)-induced Ca(2+) release channels. Hypotonic swelling was associated with a transient increase in tyrosine phosphorylation of PLCgamma, with kinetics that paralleled the increases in intracellular IP(3) levels and cytosolic [Ca(2+)]. Confocal imaging of HTC cells exposed to hypotonic medium revealed a swelling-induced association of tyrosine-phosphorylated PLCgamma with the plasma membrane. These findings suggest that activation of PLCgamma by hepatocellular swelling leads to the generation of IP(3) and stimulates discharge of Ca(2+) from the endoplasmic reticulum via activation of IP(3) receptors. By extension, these data support the concept that tyrosine phosphorylation of PLCgamma represents a critical step in adaptive responses to hepatocellular swelling.     

CD44v10 interaction with Rho-kinase (ROK) activates inositol 1,4,5-triphosphate (IP3) receptor-mediated Ca2+ signaling during hyaluronan (HA)-induced endothelial cell migration

Aortic endothelial cells (GM7372A) express a major cell adhesion molecule, CD44v10, which binds the extracellular matrix component, hyaluronan (HA), at its external domain and interacts with various signaling molecules at its cytoplasmic domain. In this study, we have determined that CD44v10 and Rho-Kinase (ROK) are physically associated as a complex in vivo. Using a recombinant fragment of ROK (in particular, the pleckstrin homology [PH] domain) and in vitro binding assays, we have detected a specific binding interaction between the PH domain of ROK and the cytoplasmic domain of CD44. Scatchard plot analysis indicates that there is a single high-affinity CD44 binding site in the PH domain of ROK with an apparent dissociation constant (Kd) of 1.76 nM, which is comparable to CD44 binding (Kd approximately 1.56 nM) to intact ROK. These findings suggest that the PH domain is the primary ROK binding region for CD44. Furthermore, HA binding to GM7372A cells promotes RhoA-mediated ROK activity, which, in turn, increases phosphorylation of three different inositol 1, 4, 5-trisphosphate receptors (IP(3)Rs) [in particular, subtype 1 (IP(3)R1), and to a lesser extent subtype 2 (IP(3)R2) and subtype 3 (IP(3)R3)] all known as IP(3)-gated Ca(2+) channels. The phosphorylated IP(3)R1 (but not IP(3)R2 or IP(3)R3) is enhanced in its binding to IP(3) which subsequently stimulates IP(3)-mediated Ca(2+) flux. Transfection of the endothelial cells with ROK's PH cDNA significantly reduces ROK association with CD44v10, and effectively inhibits ROK-mediated phosphorylation of IP(3)Rs and IP(3)R-mediated Ca(2+) flux in vitro. The PH domain of ROK also functions as a dominant-negative mutant in vivo to block HA-dependent, CD44v10-specific intracellular Ca(2+) mobilization and endothelial cell migration. Taken together, we believe that CD44v10 interaction with ROK plays a pivotal role in IP(3)R-mediated Ca(2+) signaling during HA-mediated endothelial cell migration.     

Amplification of receptor signalling by Ca2+ entry-mediated translocation and activation of PLCgamma2 in B lymphocytes

In non-excitable cells, receptor-activated Ca2+ signalling comprises initial transient responses followed by a Ca2+ entry-dependent sustained and/or oscillatory phase. Here, we describe the molecular mechanism underlying the second phase linked to signal amplification. An in vivo inositol 1,4,5-trisphosphate (IP3) sensor revealed that in B lymphocytes, receptor-activated and store-operated Ca2+ entry greatly enhanced IP3 production, which terminated in phospholipase Cgamma2 (PLCgamma2)-deficient cells. Association between receptor-activated TRPC3 Ca2+ channels and PLCgamma2, which cooperate in potentiating Ca2+ responses, was demonstrated by co-immunoprecipitation. PLCgamma2-deficient cells displayed diminished Ca2+ entry-induced Ca2+ responses. However, this defect was canceled by suppressing IP3-induced Ca2+ release, implying that IP3 and IP3 receptors mediate the second Ca2+ phase. Furthermore, confocal visualization of PLCgamma2 mutants demonstrated that Ca2+ entry evoked a C2 domain-mediated PLCgamma2 translocation towards the plasma membrane in a lipase-independent manner to activate PLCgamma2. Strikingly, Ca2+ entry-activated PLCgamma2 maintained Ca2+ oscillation and extracellular signal-regulated kinase activation downstream of protein kinase C. We suggest that coupling of Ca2+ entry with PLCgamma2 translocation and activation controls the amplification and co-ordination of receptor signalling.     

Hyaluronan-CD44 interaction with Rac1-dependent protein kinase N-gamma promotes phospholipase Cgamma1 activation, Ca(2+) signaling, and cortactin-cytoskeleton function leading to keratinocyte adhesion and differentiation

In this study we have investigated hyaluronan (HA)-CD44 interaction with protein kinase N-gamma (PKNgamma), a small GTPase (Rac1)-activated serine/threonine kinase in human keratinocytes. By using a variety of biochemical and molecular biological techniques, we have determined that CD44 and PKNgamma kinase (molecular mass approximately 120 kDa) are physically linked in vivo. The binding of HA to keratinocytes promotes PKNgamma kinase recruitment into a complex with CD44 and subsequently stimulates Rac1-mediated PKNgamma kinase activity. The Rac1-activated PKNgamma in turn increases threonine (but not serine) phosphorylation of phospholipase C (PLC) gamma1 and up-regulates PLCgamma1 activity leading to the onset of intracellular Ca(2+) mobilization. HA/CD44-activated Rac1-PKNgamma also phosphorylates the cytoskeletal protein, cortactin, at serine/threonine residues. The phosphorylation of cortactin by Rac1-PKNgamma attenuates its ability to cross-link filamentous actin in vitro. Further analyses indicate that the N-terminal antiparallel coiled-coil (ACC) domains of PKNgamma interact directly with Rac1 in a GTP-dependent manner. The binding of HA to CD44 induces PKNgamma association with endogenous Rac1 and its activity in keratinocytes. Transfection of keratinocytes with PKNgamma-ACCcDNA reduces HA-mediated recruitment of endogenous Rac1 to PKNgamma and blocks PKNgamma activity. These findings suggest that the PKNgamma-ACC fragment acts as a potent competitive inhibitor of endogenous Rac1 binding to PKNgamma in vivo. Most important, the PKNgamma-ACC fragment functions as a strong dominant-negative mutant that effectively inhibits HA/CD44-mediated PKNgamma phosphorylation of PLCgamma1 and cortactin as well as keratinocyte signaling (e.g. Ca(2+) mobilization and cortactin-actin binding) and cellular functioning (e.g. cell-cell adhesion and differentiation). Taken together, these findings strongly suggest that hyaluronan-CD44 interaction with Rac1-PKNgamma plays a pivotal role in PLCgamma1-regulated Ca(2+) signaling and cortactin-cytoskeleton function required for keratinocyte cell-cell adhesion and differentiation.     

The probability of triggering calcium puffs is linearly related to the number of inositol trisphosphate receptors in a cluster

Puffs are local Ca(2+) signals that arise by Ca(2+) liberation from the endoplasmic reticulum through concerted opening of tightly clustered inositol trisphosphate receptor/channels (IP(3)R). They serve both local signaling functions and trigger global Ca(2+) waves. The numbers of functional IP(3)R within clusters differ appreciably between different puff sites, and we investigated how the probability of puff occurrence varies with cluster size. We imaged puffs in SH-SY5Y cells using total internal fluorescence microscopy, and estimated cluster sizes from the magnitude of the largest puff observed at each site relative to the signal from a single channel. We find that the initial triggering rate of puffs following photorelease of IP(3), and the average frequency of subsequent repetitive puffs, vary about linearly with cluster size. These data accord well with stochastic simulations in which opening of any individual IP(3)R channel within a cluster triggers a puff via Ca(2+)-induced Ca(2+) release. An important consequence is that the signaling power of a puff site (average amount of Ca(2+) released per puff × puff frequency) varies about the square of cluster size, implying that large clusters contribute disproportionately to cellular signaling and, because of their higher puff frequency, preferentially act as pacemakers to initiate Ca(2+) waves.     

Molecular cloning of mouse type 2 and type 3 inositol 1,4,5-trisphosphate receptors and identification of a novel type 2 receptor splice variant

We isolated cDNAs encoding type 2 and type 3 inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)R2 and IP(3)R3, respectively) from mouse lung and found a novel alternative splicing segment, SI(m2), at 176-208 of IP(3)R2. The long form (IP(3)R2 SI(m2)(+)) was dominant, but the short form (IP(3)R2 SI(m2)(-)) was detected in all tissues examined. IP(3)R2 SI(m2)(-) has neither IP(3) binding activity nor Ca(2+) releasing activity. In addition to its reticular distribution, IP(3)R2 SI(m2)(+) is present in the form of clusters in the endoplasmic reticulum of resting COS-7 cells, and after ATP or Ca(2+) ionophore stimulation, most of the IP(3)R2 SI(m2)(+) is in clusters. IP(3)R3 is localized uniformly on the endoplasmic reticulum of resting cells and forms clusters after ATP or Ca(2+) ionophore stimulation. IP(3)R2 SI(m2)(-) does not form clusters in either resting or stimulated cells. IP(3) binding-deficient site-directed mutants of IP(3)R2 SI(m2)(+) and IP(3)R3 fail to form clusters, indicating that IP(3) binding is involved in the cluster formation by these isoforms. Coexpression of IP(3)R2 SI(m2)(-) prevents stimulus-induced IP(3)R clustering, suggesting that IP(3)R2 SI(m2)(-) functions as a negative coordinator of stimulus-induced IP(3)R clustering. Expression of IP(3)R2 SI(m2)(-) in CHO-K1 cells significantly reduced ATP-induced Ca(2+) entry, but not Ca(2+) release, suggesting that the novel splice variant of IP(3)R2 specifically influences the dynamics of the sustained phase of Ca(2+) signals.     

Timescales of IP(3)-evoked Ca(2+) spikes emerge from Ca(2+) puffs only at the cellular level

The behavior of biological systems is determined by the properties of their component molecules, but the interactions are usually too complex to understand fully how molecular behavior generates cellular behavior. Ca(2+) signaling by inositol trisphosphate receptors (IP(3)R) offers an opportunity to understand this relationship because the cellular behavior is defined largely by Ca(2+)-mediated interactions between IP(3)R. Ca(2+) released by a cluster of IP(3)R (giving a local Ca(2+) puff) diffuses and ignites the behavior of neighboring clusters (to give repetitive global Ca(2+) spikes). We use total internal reflection fluorescence microscopy of two mammalian cell lines to define the temporal relationships between Ca(2+) puffs (interpuff intervals, IPI) and Ca(2+) spikes (interspike intervals) evoked by flash photolysis of caged IP(3). We find that IPI are much shorter than interspike intervals, that puff activity is stochastic with a recovery time that is much shorter than the refractory period of the cell, and that IPI are not periodic. We conclude that Ca(2+) spikes do not arise from oscillatory dynamics of IP(3)R clusters, but that repetitive Ca(2+) spiking with its longer timescales is an emergent property of the dynamics of the whole cluster array.     

A specific role of phosphatidylinositol 3-kinase gamma. A regulation of autonomic Ca(2)+ oscillations in cardiac cells

Purinergic stimulation of cardiomyocytes turns on a Src family tyrosine kinase-dependent pathway that stimulates PLCgamma and generates IP(3), a breakdown product of phosphatidylinositol 4,5-bisphosphate (PIP2). This signaling pathway closely regulates cardiac cell autonomic activity (i.e., spontaneous cell Ca(2+) spiking). PIP2 is phosphorylated on 3' by phosphoinositide 3-kinases (PI3Ks) that belong to a broad family of kinase isoforms. The product of PI3K, phosphatidylinositol 3,4,5-trisphosphate, regulates activity of PLCgamma. PI3Ks have emerged as crucial regulators of many cell functions including cell division, cell migration, cell secretion, and, via PLCgamma, Ca(2+) homeostasis. However, although PI3Kalpha and -beta have been shown to mediate specific cell functions in nonhematopoietic cells, such a role has not been found yet for PI3Kgamma.We report that neonatal rat cardiac cells in culture express PI3Kalpha, -beta, and -gamma. The purinergic agonist predominantly activates PI3Kgamma. Both wortmannin and LY294002 prevent tyrosine phosphorylation, and membrane translocation of PLCgamma as well as IP(3) generation in ATP-stimulated cells. Furthermore, an anti-PI3Kgamma, but not an anti-PI3Kbeta, injected in the cells prevents the effect of ATP on cell Ca(2+) spiking. A dominant negative mutant of PI3Kgamma transfected in the cells also exerts the same action. The effect of ATP was observed on spontaneous Ca(2+) spiking of wild-type but not of PI3Kgamma(2/2) embryonic stem cell-derived cardiomyocytes. ATP activates the Btk tyrosine kinase, Tec, and induces its association with PLCgamma. A dominant negative mutant of Tec blocks the purinergic effect on cell Ca(2+) spiking. Tec is translocated to the T-tubes upon ATP stimulation of cardiac cells. Both an anti-PI3Kgamma antibody and a dominant negative mutant of PI3Kgamma injected or transfected into cells prevent the latter event.We conclude that PI3Kgamma activation is a crucial step in the purinergic regulation of cardiac cell spontaneous Ca(2+) spiking. Our data further suggest that Tec works in concert with a Src family kinase and PI3Kgamma to fully activate PLCgamma in ATP-stimulated cardiac cells. This cluster of kinases provides the cardiomyocyte with a tight regulation of IP(3) generation and thus cardiac autonomic activity.     

Three-dimensional distribution of cardiac Na+-Ca2+ exchanger and ryanodine receptor during development

Mechanisms of cardiac excitation-contraction coupling in neonates are still not clearly defined. Previous work in neonates shows reverse-mode Na(+)-Ca(2+) exchange to be the primary route of Ca(2+) entry during systole and the neonatal sarcoplasmic reticulum to have similar capability as that of adult in storing and releasing Ca(2+). We investigated Na(+)-Ca(2+) exchanger (NCX) and ryanodine receptor (RyR) distribution in developing ventricular myocytes using immunofluorescence, confocal microscopy, and digital image analysis. In neonates, both NCX and RyR clusters on the surface of the cell displayed a short longitudinal periodicity of approximately 0.7 microm. However, by adulthood, both proteins were also found in the interior. In the adult, clusters of NCX on the surface of the cell retained the approximately 0.7-microm periodicity whereas clusters of RyR adopted a longer longitudinal periodicity of approximately 2.0 microm. This suggests that neonatal myocytes also have a peri-M-line RyR distribution that is absent in adult myocytes. NCX and RyR colocalized voxel density was maximal in neonates and declined significantly with ontogeny. We conclude in newborns, Ca(2+) influx via NCX could potentially activate the dense network of peripheral Ca(2+) stores via peripheral couplings, evoking Ca(2+)-induced Ca(2+) release.     

Binding of cytoplasmic proteins to the CD19 intracellular domain is high affinity, competitive, and multimeric

CD19 is required for the development of B1 and marginal zone B cells, for Ab responses, and for B cell memory. CD19 immunoprecipitates contain a complex of cytoplasmic proteins, including Lyn, Vav, phospholipase Cgamma2 (PLCgamma2), Grb2, and the p85 subunit of phosphatidylinositol 3-kinase. Which of these bind directly to CD19 and the strengths of the interactions are unknown. These issues are important in understanding the signaling functions of CD19, which are crucial for normal B cell physiology. Using purified, recombinant proteins, we now show that each of these signaling proteins contains at least one Src homology 2 (SH2) domain that interacts directly with the phosphorylated CD19 cytoplasmic domain. The affinities of binding of the SH2 domains of Vav, p85, and Grb2 to CD19 are each in the nanomolar range by surface plasmon resonance (Biacore) analysis. Binding of Lyn and PLCgamma2 do not fit 1:1 modeling. However, analyses of binding data (Lyn) and competition experiments (PLCgamma2) suggest that these bind with comparable affinity. Competition experiments demonstrate that SH2 domains whose binding is dependent on the same CD19 tyrosine(s) compete for binding, but these SH2 domains do not impede binding of different SH2 domains to other CD19 tyrosines. We conclude that binding to the CD19 cytoplasmic domain is multimeric, high affinity, and competitive. The high affinity of the interactions also suggests that tyrosines that were nonessential in vivo are nevertheless functional. A preliminary structural model suggests that CD19 forms a signaling complex containing multiple cytoplasmic proteins in close proximity to each other and to the plasma membrane.     

High resolution mapping of mast cell membranes reveals primary and secondary domains of Fc(epsilon)RI and LAT

In mast cells, cross-linking the high-affinity IgE receptor (Fc(epsilon)RI) initiates the Lyn-mediated phosphorylation of receptor ITAMs, forming phospho-ITAM binding sites for Syk. Previous immunogold labeling of membrane sheets showed that resting Fc(epsilon)RI colocalize loosely with Lyn, whereas cross-linked Fc(epsilon)RI redistribute into specialized domains (osmiophilic patches) that exclude Lyn, accumulate Syk, and are often bordered by coated pits. Here, the distribution of Fc(epsilon)RI beta is mapped relative to linker for activation of T cells (LAT), Grb2-binding protein 2 (Gab2), two PLCgamma isoforms, and the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase), all implicated in the remodeling of membrane inositol phospholipids. Before activation, PLCgamma1 and Gab2 are not strongly membrane associated, LAT occurs in small membrane clusters separate from receptor, and PLCgamma2, that coprecipitates with LAT, occurs in clusters and along cytoskeletal cables. After activation, PLCgamma2, Gab2, and a portion of p85 colocalize with Fc(epsilon)RI beta in osmiophilic patches. LAT clusters enlarge within 30 s of receptor activation, forming elongated complexes that can intersect osmiophilic patches without mixing. PLCgamma1 and another portion of p85 associate preferentially with activated LAT. Supporting multiple distributions of PI3-kinase, Fc(epsilon)RI cross-linking increases PI3-kinase activity in anti-LAT, anti-Fc(epsilon)RIbeta, and anti-Gab2 immune complexes. We propose that activated mast cells propagate signals from primary domains organized around Fc(epsilon)RIbeta and from secondary domains, including one organized around LAT.     

Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa and phospholipase C gamma 1 are required for NF-kappa B activation and lipid raft recruitment of protein kinase C theta induced by T cell costimulation

The NF-kappaB activation pathway induced by T cell costimulation uses various molecules including Vav1 and protein kinase C (PKC)theta. Because Vav1 inducibly associates with further proteins including phospholipase C (PLC)gamma1 and Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76), we investigated their role for NF-kappaB activation in Jurkat leukemia T cell lines deficient for expression of these two proteins. Cells lacking SLP-76 or PLCgamma1 failed to activate NF-kappaB in response to T cell costimulation. In contrast, replenishment of SLP-76 or PLCgamma1 expression restored CD3/CD28-induced IkappaB kinase (IKK) activity as well as NF-kappaB DNA binding and transactivation. PKCtheta activated NF-kappaB in SLP-76- and PLCgamma1-deficient cells, showing that PKCtheta is acting further downstream. In contrast, Vav1-induced NF-kappaB activation was normal in SLP-76(-) cells, but absent in PLCgamma1(-) cells. CD3/CD28-stimulated recruitment of PKCtheta and IKKgamma to lipid rafts was lost in SLP-76- or PLCgamma1-negative cells, while translocation of Vav1 remained unaffected. Accordingly, recruitment of PKCtheta to the immunological synapse strictly relied on the presence of SLP-76 and PLCgamma1, but synapse translocation of Vav1 identified in this study was independent from both proteins. These results show the importance of SLP-76 and PLCgamma1 for NF-kappaB activation and raft translocation of PKCtheta and IKKgamma.     

Evidence that phospholipase C from the sperm is not responsible for initiating Ca(2+) release at fertilization in mouse eggs

Release of Ca(2+) from intracellular stores at fertilization of mammalian eggs is mediated by inositol 1,4,5-trisphosphate (IP3), but the mechanism by which the sperm initiates IP3 production is not yet understood. We tested the hypothesis that phospholipase C (PLC) activity introduced into the mouse egg as a consequence of sperm-egg fusion is responsible for causing Ca(2+) release. We demonstrated that microinjecting purified, recombinant PLCgamma1 protein into mouse eggs caused Ca(2+) oscillations like those seen at fertilization. However, the PLC activity in the minimum amount of purified PLCgamma1 protein needed to elicit Ca(2+) release when injected into eggs was approximately 500-900 times the PLC activity contained in a single sperm. This indicates that a single mouse sperm does not contain enough PLC activity to be responsible for causing Ca(2+) release at fertilization. We also examined whether phosphatidylinositol 3-kinase (PI3K) could have a role in this process, and found that several inhibitors of PI3K-mediated signaling had no effect on Ca(2+) release at fertilization.     

CD22 regulates B cell receptor-mediated signals via two domains that independently recruit Grb2 and SHP-1

Recognition of antigen by the B cell antigen receptor (BCR) determines the subsequent fate of a B cell and is regulated in part by the involvement of other surface molecules, termed coreceptors. CD22 is a B cell-restricted coreceptor that gets rapidly tyrosyl-phosphorylated and recruits various signaling molecules to the membrane following BCR ligation. Although CD22 contains three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), only the two carboxyl-terminal ITIM tyrosines are required for efficient recruitment of the SHP-1 phosphatase after BCR ligation. Furthermore, Grb2 is inducibly recruited to CD22 in human and murine B cells. Unlike SHP-1, Grb2 recruitment to CD22 is not inhibited by specific doses of the Src family kinase-specific inhibitor PP1. The tyrosine residue in CD22 required for Grb2 recruitment (Tyr-828) is distinct and independent from the two ITIM tyrosines required for efficient SHP-1 recruitment (Tyr-843 and Tyr-863). Individually both Lyn and Syk are required for maximal phosphorylation of CD22 following ligation of the BCR, and together Lyn and Syk are required for all of the constitutive and induced tyrosine phosphorylation of CD22. We propose that the cytoplasmic tail of CD22 contains two domains that regulate signal transduction pathways initiated by the BCR and B cell fate.     

TRPC3 controls agonist-stimulated intracellular Ca2+ release by mediating the interaction between inositol 1,4,5-trisphosphate receptor and RACK1

Activation of TRPC3 channels is concurrent with inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R)-mediated intracellular Ca(2+) release and associated with phosphatidylinositol 4,5-bisphosphate hydrolysis and recruitment to the plasma membrane. Here we report that interaction of TRPC3 with receptor for activated C-kinase-1 (RACK1) not only determines plasma membrane localization of the channel but also the interaction of IP(3)R with RACK1 and IP(3)-dependent intracellular Ca(2+) release. We show that TRPC3 interacts with RACK1 via N-terminal residues Glu-232, Asp-233, Glu-240, and Glu-244. Carbachol (CCh) stimulation of HEK293 cells expressing wild type TRPC3 induced recruitment of a ternary TRPC3-RACK1-IP(3)R complex and increased surface expression of TRPC3 and Ca(2+) entry. Mutation of the putative RACK1 binding sequence in TRPC3 disrupted plasma membrane localization of the channel. CCh-stimulated recruitment of TRPC3-RACK1-IP(3)R complex as well as increased surface expression of TRPC3 and receptor-operated Ca(2+) entry were also attenuated. Importantly, CCh-induced intracellular Ca(2+) release was significantly reduced as was RACK1-IP(3)R association without any change in thapsigargin-stimulated Ca(2+) release and entry. Knockdown of endogenous TRPC3 also decreased RACK1-IP(3)R association and decreased CCh-stimulated Ca(2+) entry. Furthermore, an oscillatory pattern of CCh-stimulated intracellular Ca(2+) release was seen in these cells compared with the more sustained pattern seen in control cells. Similar oscillatory pattern of Ca(2+) release was seen after CCh stimulation of cells expressing the TRPC3 mutant. Together these data demonstrate a novel role for TRPC3 in regulation of IP(3)R function. We suggest TRPC3 controls agonist-stimulated intracellular Ca(2+) release by mediating interaction between IP(3)R and RACK1.     

Inositol (1,4,5)-trisphosphate receptor microarchitecture shapes Ca2+ puff kinetics

Inositol (1,4,5)-trisphosphate receptors (IP(3)Rs) release intracellular Ca(2+) as localized Ca(2+) signals (Ca(2+) puffs) that represent the activity of small numbers of clustered IP(3)Rs spaced throughout the endoplasmic reticulum. Although much emphasis has been placed on estimating the number of active Ca(2+) release channels supporting Ca(2+) puffs, less attention has been placed on understanding the role of cluster microarchitecture. This is important as recent data underscores the dynamic nature of IP(3)R transitions between heterogeneous cellular architectures and the differential behavior of IP(3)Rs socialized into clusters. Here, we applied a high-resolution model incorporating stochastically gating IP(3)Rs within a three-dimensional cytoplasmic space to demonstrate: 1), Ca(2+) puffs are supported by a broad range of clustered IP(3)R microarchitectures; 2), cluster ultrastructure shapes Ca(2+) puff characteristics; and 3), loosely corralled IP(3)R clusters (>200 nm interchannel separation) fail to coordinate Ca(2+) puffs, owing to inefficient triggering and impaired coupling due to reduced Ca(2+)-induced Ca(2+) release microwave velocity (<10 nm/s) throughout the channel array. Dynamic microarchitectural considerations may therefore influence Ca(2+) puff occurrence/properties in intact cells, contrasting with a more minimal role for channel number over the same simulated conditions in shaping local Ca(2+) dynamics.     

TRPC3 is the erythropoietin-regulated calcium channel in human erythroid cells

Erythropoietin (Epo) stimulates a significant increase in the intracellular calcium concentration ([Ca(2+)](i)) through activation of the murine transient receptor potential channel TRPC2, but TRPC2 is a pseudogene in humans. TRPC3 expression increases on normal human erythroid progenitors during differentiation. Here, we determined that erythropoietin regulates calcium influx through TRPC3. Epo stimulation of HEK 293T cells transfected with Epo receptor and TRPC3 resulted in a dose-dependent increase in [Ca(2+)](i), which required extracellular calcium influx. Treatment with the phospholipase C (PLC) inhibitor U-73122 or down-regulation of PLCgamma1 by RNA interference inhibited the Epo-stimulated increase in [Ca(2+)](i) in TRPC3-transfected HEK 293T cells and in primary human erythroid precursors, demonstrating a requirement for PLC. TRPC3 associated with PLCgamma, and substitution of predicted PLCgamma Src homology 2 binding sites (Y226F, Y555F, Y648F, and Y674F) on TRPC3 reduced the interaction of TRPC3 with PLCgamma and inhibited the rise in [Ca(2+)](i). Substitution of Tyr(226) alone with phenylalanine significantly reduced the Epo-stimulated increase in [Ca(2+)](i) but not the association of PLCgamma with TRPC3. PLC activation results in production of inositol 1,4,5-trisphosphate (IP(3)). To determine whether IP(3) is involved in Epo activation of TRPC3, TRPC3 mutants were prepared with substitution or deletion of COOH-terminal IP(3) receptor (IP(3)R) binding domains. In cells expressing TRPC3 with mutant IP(3)R binding sites and Epo receptor, interaction of IP(3)R with TRPC3 was abolished, and Epo-modulated increase in [Ca(2+)](i) was reduced. Our data demonstrate that Epo modulates TRPC3 activation through a PLCgamma-mediated process that requires interaction of PLCgamma and IP(3)R with TRPC3. They also show that TRPC3 Tyr(226) is critical in Epo-dependent activation of TRPC3. These data demonstrate a redundancy of TRPC channel activation mechanisms by widely different agonists.