The regulation of luteinizing hormone exocytosis in α-toxin permeabilized sheep anterior pituitary cells

Although exocytosis is the major mechanism by which cells secrete products into their environment, little is known about the mechanism of this fundamental process. Previous studies on the regulation of luteinizing hormone (LH) exocytosis have used intact cells exclusively. It is not possible, however, to determine the precise requirements for exocytosis in intact cells since the cytosol is not directly accessible. Permeabilization of the plasma membrane allows experimental manipulation of the intracellular milieu while preserving the exocytic apparatus. The diameter of the atoxin pores (2-3 nm) allowed the exchange of small molecules such as ATP while larger cytosolic proteins such as lactate dehydrogenase were retained. Because of the slow exchange of small molecules through a-toxin pores a protocol was developed which combines prolonged pre-equilibration of the permeabilized cells at 0°C before stimulation with strong Ca²⁺ buffering. Under these conditions an increase in the [Ca²⁺]free stimulated a 15-20 fold increase in LH exocytosis (EC₅₀ pCa 5.5). After 12-15 minutes the rate of exocytosis declined and the cells became refractory to Ca²⁺. At resting [Ca²⁺]free (pea 7), cAMP stimulated a rapid, 2 - 3 fold, increase in LH exocytosis. cAMP caused a modest enhancement of Ca²⁺-stimulated LH exocytosis by causing a left shift in the EC₅₀ for Ca²⁺ from pCa 5.6 to pCa 5.9. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) synergistically enhanced cAMP-stimulated LH exocytosis, an effect which was further augmented by increasing the [Ca²⁺]free· Gonadotrophin-releasing hormone (GnRH) was found to stimulate cAMP production in intact pituitary cells. Since previous studies have shown that GnRH activates PKC and stimulates a rise in cytosolic [Ca²⁺]free, these results suggest that a synergistic interaction of the cAMP, PKC and Ca²⁺ second messenger systems is of importance in the mechanism of GnRH-stimulated LH exocytosis. When permeabilized cells were equilibrated for prolonged periods in the absence of MgATP, Ca²⁺-stimulated LH exocytosis declined. The time course of the decline closely followed the leakage of intracellular ¹⁴C-ATP. Addition of MgATP rapidly restored full Ca²⁺-stimulated LH exocytosis. Ca²⁺-, cAMP-, and PMA-stimulated LH exocytosis were all dependent on millimolar MgATP concentrations (EC₅₀ 1 .5-3 mM). It has been postulated that PKC is a mediator of Ca²⁺- stimulated exocytosis. Several findings in the present study argue against this hypothesis. Firstly, PMA and Ca²⁺ had additive effects on LH exocytosis at all [Ca²⁺]free· Secondly, PMA was able to stimulate further LH release from cells made refractory to high [Ca²⁺]free· Thirdly, the PKC inhibitor staurosporine did not inhibit Ca²⁺-stimulated LH exocytosis under conditions in which it inhibited PMAstimulated exocytosis. Fourthly, in cells desensitized to PMA by prolonged exposure to a high PMA concentrations, Ca²⁺-stimulated LH exocytosis was not inhibited. And finally, Ba²⁺+ was able to stimulate LH exocytosis to a maximal extent similar to Ca²⁺ despite the fact that Ba²⁺+ is an extremely poor activator of PKC. Since Ba²⁺+ is also a poor activator of calmodulin, this latter result implies that calmodulin does not mediate the effect of Ca²⁺. In agreement with this, the calmodulin inhibitor calmidazolium did not inhibit Ca²⁺-stimulated LH exocytosis. Since GTP-binding proteins have been implicated in regulated exocytosis in other cell systems, the effects of guanine nucleotides on LH exocytosis were examined. At resting cytosolic [Ca²⁺]free (pea 7), the GTP analogues GTPyS and GMPPNP stimulated LH exocytosis with similar potencies (EC₅₀ 20-50 μM). Additional experiments indicated that the effects of these GTP analogues could not be explained by activation of either PKC alone or cAMP-dependent protein kinase alone. In the presence of both PMA and cAMP, GMPPNP did not stimulate a further increase in the rate of LH exocytosis, suggesting that the stimulatory actions of guanine nucleotides may be mediated by the combined activation of PKC and generation of cAMP, as a result of activation of signal-transducing G proteins. In contrast, pretreatment of cells with GTPyS at low [Ca²⁺]free markedly inhibited subsequent responses to Ca²⁺, cAMP, PMA, and cAMP plus PMA. This inhibitory effect required lower GTPyS concentrations than the stimulatory effect (IC₅₀ 1-10 μM), and was not observed with GMPPNP. These findings indicate the involvement of a distinct guanine nucleotide-binding protein in exocytosis at a site distal to second messenger generation

Multisite Phosphorylation Modulates the T Cell Receptor ζ-Chain Potency but not the Switchlike Response

AbstractMultisite phosphorylation is ubiquitous in cellular signaling and is thought to provide signaling proteins with additional regulatory mechanisms. Indeed, mathematical models have revealed a large number of mechanisms by which multisite phosphorylation can produce switchlike responses. The T cell antigen receptor (TCR) is a multisubunit receptor on the surface of T cells that is a prototypical multisite substrate as it contains 20 sites that are distributed on 10 conserved immunoreceptor tyrosine-based activation motifs (ITAMs). The TCR ζ-chain is a homodimer subunit that contains six ITAMs (12 sites) and exhibits a number of properties that are predicted to be sufficient for a switchlike response. We have used cellular reconstitution to systematically study multisite phosphorylation of the TCR ζ-chain. We find that multisite phosphorylation proceeds by a nonsequential random mechanism, and find no evidence that multiple ITAMs modulate a switchlike response but do find that they alter receptor potency and maximum phosphorylation. Modulation of receptor potency can be explained by a reduction in molecular entropy of the disordered ζ-chain upon phosphorylation. We further find that the tyrosine kinase ZAP-70 increases receptor potency but does not modulate the switchlike response. In contrast to other multisite proteins, where phosphorylations act in strong concert to modulate protein function, we suggest that the multiple ITAMs on the TCR function mainly to amplify subsequent signaling

Matched sizes of activating and inhibitory receptor/ligand pairs are required for optimal signal integration by human Natural Killer cells

It has been suggested that receptor-ligand complexes segregate or co-localise within immune synapses according to their size, and this is important for receptor signaling. Here, we set out to test the importance of receptor-ligand complex dimensions for immune surveillance of target cells by human Natural Killer (NK) cells. NK cell activation is regulated by integrating signals from activating receptors, such as NKG2D, and inhibitory receptors, such as KIR2DL1. Elongating the NKG2D ligand MICA reduced its ability to trigger NK cell activation. Conversely, elongation of KIR2DL1 ligand HLA-C reduced its ability to inhibit NK cells. Whereas normal-sized HLA-C was most effective at inhibiting activation by normal-length MICA, only elongated HLA-C could inhibit activation by elongated MICA. Moreover, HLA-C and MICA that were matched in size co-localised, whereas HLA-C and MICA that were different in size were segregated. These results demonstrate that receptor-ligand dimensions are important in NK cell recognition, and suggest that optimal integration of activating and inhibitory receptor signals requires the receptor-ligand complexes to have similar dimensions

Systems model of T cell receptor proximal signaling reveals emergent ultrasensitivity

Receptor phosphorylation is thought to be tightly regulated because phosphorylated receptors initiate signaling cascades leading to cellular activation. The T cell antigen receptor (TCR) on the surface of T cells is phosphorylated by the kinase Lck and dephosphorylated by the phosphatase CD45 on multiple immunoreceptor tyrosine-based activation motifs (ITAMs). Intriguingly, Lck sequentially phosphorylates ITAMs and ZAP-70, a cytosolic kinase, binds to phosphorylated ITAMs with differential affinities. The purpose of multiple ITAMs, their sequential phosphorylation, and the differential ZAP-70 affinities are unknown. Here, we use a systems model to show that this signaling architecture produces emergent ultrasensitivity resulting in switch-like responses at the scale of individual TCRs. Importantly, this switch-like response is an emergent property, so that removal of multiple ITAMs, sequential phosphorylation, or differential affinities abolishes the switch. We propose that highly regulated TCR phosphorylation is achieved by an emergent switch-like response and use the systems model to design novel chimeric antigen receptors for therapy

TCR-pMHC kinetics under force in a cell-free system show no intrinsic catch bond, but a minimal encounter duration before binding

International audienceThe TCR-pMHC interaction is the only antigen specific interaction during T lymphocyte activation. Recent work suggests that formation of catch bonds is characteristic of activating TCR-pMHC interactions. However, whether this binding behavior is an intrinsic feature of the molecular bond, or a consequence of more complex multimolecular or cellular responses, remains unclear. We used a laminar flow chamber to measure, firstly, 2D TCR-pMHC dissociation kinetics of peptides of various activating potency in a cell-free system in the force range (6-15pN) previously associated with catch-slip transitions and, secondly, 2D TCR-pMHC association kinetics, for which the method is well-suited. We did not observe catch bonds in dissociation, and the off-rate measured in the 6-15pN range correlated well with activation potency, suggesting that formation of catch bonds is not an intrinsic feature of the TCR-pMHC interaction. The association kinetics were better explained by a model with a minimal encounter duration rather than a standard on-rate constant, suggesting that membrane fluidity and dynamics may strongly influence bond formation

Systems model of T cell receptor proximal signaling reveals emergent ultrasensitivity. PLoS Comput Biol (2013) 9:e1003004. doi:10.1371/journal.pcbi.1003004 54. Wu Y, Vendome J, Shapiro L, Ben-Shaul A, Honig B. Transforming binding affinities from three di

Abstract Receptor phosphorylation is thought to be tightly regulated because phosphorylated receptors initiate signaling cascades leading to cellular activation. The T cell antigen receptor (TCR) on the surface of T cells is phosphorylated by the kinase Lck and dephosphorylated by the phosphatase CD45 on multiple immunoreceptor tyrosine-based activation motifs (ITAMs). Intriguingly, Lck sequentially phosphorylates ITAMs and ZAP-70, a cytosolic kinase, binds to phosphorylated ITAMs with differential affinities. The purpose of multiple ITAMs, their sequential phosphorylation, and the differential ZAP-70 affinities are unknown. Here, we use a systems model to show that this signaling architecture produces emergent ultrasensitivity resulting in switch-like responses at the scale of individual TCRs. Importantly, this switch-like response is an emergent property, so that removal of multiple ITAMs, sequential phosphorylation, or differential affinities abolishes the switch. We propose that highly regulated TCR phosphorylation is achieved by an emergent switch-like response and use the systems model to design novel chimeric antigen receptors for therapy

ZAP-70 binding to phosphorylated ITAMs enhances both ultrasensitivity and potency.

<p>A) The concentration of total -chain phosphorylation as a function of the relative concentration of active kinase (E) to phosphatase (F). <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003004#s2" target="_blank">Results</a> are shown for sequential phosphorylation (blue, green) and random phosphorylation (red, orange) in the absence (blue, red) and presence (green, orange) of ZAP-70. B) Hill numbers and C) for all four curves reveal that ZAP-70 binding dramatically increases both ultrasensitivity and potency when phosphorylation is sequential but not random.</p

Differential ZAP-70 affinity and sequential phosphorylation produces ultrasensitivity.

<p>A) The concentration of bound ZAP-70 as a function of the relative concentration of active kinase (E) to phosphatase (F). Shown are the wild-type -chain (123), and additional constructs where all ITAMs are identical (222), switched (321), or where phosphorylation is no longer sequential (123 Random). The Hill numbers (inset) reveal that ultrasensitivity is decreased if ZAP-70 does not exhibit differential affinity (222), if the affinity decreases as the is sequentially phosphorylated (321), or if phosphorylation is no longer sequential. B) Heat map of Hill numbers as a function of the ZAP-70 unbinding rate for ITAM 1 (Z) and ITAM 3 (Z), where the unbinding rate for ITAM 2 is fixed at 1 s⁻¹. The calculation is performed under sequential phosphorylation. Maximum sensitivity is found in the top left of the heat map, where ZAP-70 binds with the largest affinity to ITAM 1 and with lowest affinity to ITAM 3. The heat map is repeated using an alternate measure of sensitivity in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003004#pcbi.1003004.s003" target="_blank">Fig. S3</a>. Note that the heat map colour scheme in panel B is not related to the colour scheme in panel A.</p

Schematic of the T cell receptor proximal signaling molecules considered in the systems model.

<p>We consider the TCR-chain containing three ITAMs, labelled as 3 (membrane-distal) to 1 (membrane-proximal). These ITAMs are sequentially phosphorylated by the tyrosine kinase Lck and dephosphorylated by the phosphatase CD45. The cytosolic kinase ZAP-70 contains tandem SH2 domains which are able to bind to doubly (fully) phosphorylated ITAMs with differential affinities, with the smallest affinity to 3 and largest affinity to 1. When bound to phosphorylated ITAMs, ZAP-70 is able to propagate signaling by phosphorylating downstream signaling molecules and adaptors.</p

The absolute ZAP-70 affinity for -chain ITAMs modulates potency.

<p>A) The concentration of bound ZAP-70 as a function of the concentration of active kinase (E) to phosphatase (F). <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003004#s2" target="_blank">Results</a> are shown for the wild-type -chain (123) and for three additional -chains that contain all high affinity ITAM 1 (111), intermediate affinity ITAM 2 (222), or low affinity ITAM 3 (333). Comparison of these -chains reveals that B) sensitivity is unchanged whilst C) potency is modulated.</p