scholarly journals Determinants of adenophostin A binding to inositol trisphosphate receptors

2002 ◽  
Vol 367 (1) ◽  
pp. 113-120 ◽  
Author(s):  
Stephen A. MORRIS ◽  
Edmund P. NEROU ◽  
Andrew M. RILEY ◽  
Barry V.L. POTTER ◽  
Colin W. TAYLOR
Keyword(s):  
Binding Domain ◽  
Sf9 Cells ◽  
Receptor Subtypes ◽  
Ip3 Receptors ◽  
Ip3 Receptor ◽  
High Affinity ◽  
Recombinant Type ◽  
Adenophostin A ◽  
Binding Core

Inositol 1,4,5-trisphosphate (IP3) receptors from cerebellum and recombinant type 1 IP3 receptors expressed in Sf9 cells had indistinguishable affinities for IP3 (Kd = 6.40±0.48nM) and adenophostin A (Kd = 0.89±0.05nM). In cytosol-like medium, each of the three mammalian IP3 receptor subtypes when expressed in Sf9 cells bound adenophostin A with greater affinity than IP3. It has been suggested that adenophostin A binds with high affinity only in the presence of ATP, but we found that adenophostin A similarly displaced [3H]IP3 from type 1 IP3 receptors whatever the ATP concentration. N-terminal fragments of the type 1 receptor were expressed with and without the S1 splice site; its removal had no effect on [3H]IP3 binding to the 1—604 protein, but abolished binding to the 224—604 protein. The 1—604 fragment and full-length receptor bound adenophostin A with the same affinity, but the fragment had 3-fold greater affinity for IP3, suggesting that C-terminal residues selectively inhibit IP3 binding. The 224—604S1+ fragment bound IP3 and adenophostin A with increased affinity, but as with the 1—604 fragment it bound adenophostin A with only 2-fold greater affinity than IP3. High-affinity binding of adenophostin A may be partially determined by its 2′-phosphate interacting more effectively than the 1-phosphate of IP3 with residues within the IP3-binding core. This may account for the 2-fold greater affinity of adenophostin A relative to IP3 for the minimal IP3-binding domain. In addition we suggest that C-terminal residues, which impede access of IP3, may selectively interact with adenophostin A to allow it unhindered access to the IP3-binding domain.

scholarly journals Differential regulation of types-1 and -3 inositol trisphosphate receptors by cytosolic Ca2+

1997 ◽  
Vol 328 (3) ◽  
pp. 785-793 ◽  
Author(s):  
Thomas J. A. CARDY ◽  
David TRAYNOR ◽  
W. Colin TAYLOR
Keyword(s):  
Inositol Trisphosphate ◽  
Receptor Subtype ◽  
Sf9 Cells ◽  
Receptor Subtypes ◽  
Ip3 Receptors ◽  
Ip3 Receptor ◽  
Single Mechanism ◽  
Receptor Isoforms ◽  
Type 3

Biphasic regulation of inositol trisphosphate (IP3)-stimulated Ca2+ mobilization by cytosolic Ca2+ is believed to contribute to regenerative intracellular Ca2+ signals. Since cells typically express several IP3 receptor isoforms and the effects of cytosolic Ca2+ are not mediated by a single mechanism, it is important to resolve the properties of each receptor subtype. Full-length rat types-1 and -3 IP3 receptors were expressed in insect Sf9 cells at levels 10-40-fold higher than the endogenous receptors. The expressed receptors were glycosylated and assembled into tetramers, and binding of [3H]IP3 to each subtype was regulated by cytosolic Ca2+. The effects of increased [Ca2+] on native cerebellar and type-1 receptors expressed in Sf9 cells were indistinguishable. A maximally effective increase in [Ca2+] reversibly inhibited [3H]IP3 binding by approx. 50% by decreasing the number of IP3-binding sites (Bmax) without affecting their affinity for IP3. The effects of Ca2+ on type-3 receptors were more complex: increasing [Ca2+] first stimulated [3H]IP3 binding by increasing Bmax, and then inhibited it by causing a substantial decrease in the affinity of the receptor for IP3. The different effects of Ca2+ on the receptor subtypes were not a consequence of limitations in the availability of accessory proteins or of artifactual effects of Ca2+ on membrane structure. We conclude that Ca2+ can inhibit IP3 binding to types-1 and -3 IP3 receptors although by different mechanisms, and that IP3 binding to type-3 receptors is stimulated at intermediate [Ca2+]. A consequence of these differences is that, at resting cytosolic [Ca2+], type-3 receptors are more sensitive than type-1 receptors to IP3, but the situation reverses at higher cytosolic [Ca2+]. Such differences may be important in generating the spatially and temporally complex changes in cytosolic [Ca2+] evoked by receptors linked to IP3 formation.

Functional properties of Drosophila inositol trisphosphate receptors

2001 ◽  
Vol 359 (2) ◽  
pp. 435-441 ◽  
Author(s):  
Jane E. SWATTON ◽  
Stephen A. MORRIS ◽  
Frank WISSING ◽  
Colin W. TAYLOR
Keyword(s):  
Functional Properties ◽  
Inositol Trisphosphate ◽  
Stable State ◽  
Hill Coefficient ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
S2 Cells ◽  
Ip3 Receptors ◽  
Ip3 Receptor ◽  
Adenophostin A

The functional properties of the only inositol trisphosphate (IP3) receptor subtype expressed in Drosophila were examined in permeabilized S2 cells. The IP3 receptors of S2 cells bound (1,4,5)IP3 with high affinity (Kd = 8.5±1.1nM), mediated positively co-operative Ca2+ release from a thapsigargin-sensitive Ca2+ store (EC50 = 75±4nM, Hill coefficient = 2.1±0.2), and they were recognized by an antiserum to a peptide conserved in all IP3 receptor subtypes in the same way as mammalian IP3 receptors. As with mammalian IP3 receptors, (2,4,5)IP3 (EC50 = 2.3±0.3μM) and (4,5)IP2 (EC50 approx. 10μM) were approx. 20- and 100-fold less potent than (1,4,5)IP3. Adenophostin A, which is typically approx. 10-fold more potent than IP3 at mammalian IP3 receptors, was 46-fold more potent than IP3 in S2 cells (EC50 = 1.67±0.07nM). Responses to submaximal concentrations of IP3 were quantal and IP3-evoked Ca2+ release was biphasically regulated by cytosolic Ca2+. Using rapid superfusion to examine the kinetics of IP3-evoked Ca2+ release from S2 cells, we established that IP3 (10μM) maximally activated Drosophila IP3 receptors within 400ms. The activity of the receptors then slowly decayed (t1/2 = 2.03±0.07s) to a stable state which had 47±1% of the activity of the maximally active state. We conclude that the single subtype of IP3 receptor expressed in Drosophila has similar functional properties to mammalian IP3 receptors and that analyses of IP3 receptor function in this genetically tractable organism are therefore likely to contribute to understanding the roles of mammalian IP3 receptors.

Selective recognition of inositol phosphates by subtypes of the inositol trisphosphate receptor

2001 ◽  
Vol 355 (1) ◽  
pp. 59-69 ◽  
Author(s):  
Edmund P. NEROU ◽  
Andrew M. RILEY ◽  
Barry V. L. POTTER ◽  
Colin W. TAYLOR
Keyword(s):  
Hydroxy Group ◽  
Inositol Phosphates ◽  
Inositol Trisphosphate ◽  
Receptor Subtypes ◽  
Ip3 Receptors ◽  
Ip3 Receptor ◽  
Subtype Selectivity ◽  
Type 3

Synthetic analogues of inositol trisphosphate (IP3), all of which included structures equivalent to the 4,5-bisphosphate of (1,4,5)IP3, were used to probe the recognition properties of rat full-length type 1, 2 and 3 IP3 receptors expressed in insect Spodoptera frugiperda 9 cells. Using equilibrium competition binding with [3H](1,4,5)IP3 in Ca2+-free cytosol-like medium, the relative affinities of the receptor subtypes for (1,4,5)IP3 were type 3 (Kd = 11±2nM)>type 2 (Kd = 17±2nM) > type 1 (Kd = 24±4nM). (1,4,5)IP3 binding was reversibly stimulated by increased pH, but the subtypes differed in their sensitivity to pH (type 1 > type 2>type 3). For all three subtypes, the equatorial 6-hydroxy group of (1,4,5)IP3 was essential for high-affinity binding, the equatorial 3-hydroxy group significantly improved affinity, and the axial 2-hydroxy group was insignificant; a 1-phosphate (or in its absence, a 2-phosphate) improved binding affinity. The subtypes differed in the extents to which they tolerated inversion of the 3-hydroxy group of (1,4,5)IP3 (type 1>type 2>type 3), and this probably accounts for the selectivity of (1,4,6)IP3 for type 1 receptors. They also differed in their tolerance of inversion, removal or substitution (by phosphate) of the 2-hydroxy group (types 2 and 3>type 1), hence the selectivity of (1,2,4,5)IP4 for type 2 and 3 receptors. Removal of the 3-hydroxy group or its replacement by fluorine or CH2OH was best tolerated by type 3 receptors, and accounts for the selectivity of 3-deoxy(1,4,5)IP3 for type 3 receptors. Our results provide the first systematic analysis of the recognition properties of IP3 receptor subtypes and have identified the 2- and 3-positions of (1,4,5)IP3 as key determinants of subtype selectivity.

scholarly journals Ca2+ and calmodulin differentially modulate myo-inositol 1,4,5-trisphosphate (IP3)-binding to the recombinant ligand-binding domains of the various IP3 receptor isoforms

2000 ◽  
Vol 346 (2) ◽  
pp. 275-280 ◽  
Author(s):  
Sara VANLINGEN ◽  
Henk SIPMA ◽  
Patrick DE SMET ◽  
Geert CALLEWAERT ◽  
Ludwig MISSIAEN ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Recombinant Proteins ◽  
Ip3 Receptor ◽  
Binding Characteristics ◽  
Binding Domains ◽  
Receptor Isoforms ◽  
Inositol 1,4,5 Trisphosphate ◽  
Adenophostin A ◽  
Trisphosphate Receptor

We have expressed the N-terminal 581 amino acids of type 1 myo-inositol 1,4,5-trisphosphate receptor (IP3R1), IP3R2 and IP3R3 as recombinant proteins [ligand-binding site 1 (lbs-1), lbs-2, lbs-3] in the soluble fraction of Escherichia coli. These recombinant proteins contain the complete IP3-binding domain and bound IP3 and adenophostin A with high affinity. Ca2+ and calmodulin were previously found to maximally inhibit IP3 binding to lbs-1 by 42±6 and 43±6% respectively, and with an IC50 of approx. 200 nM and 3 μM respectively [Sipma, De Smet, Sienaert, Vanlingen, Missiaen, Parys and De Smedt (1999) J. Biol. Chem. 274, 12157-12562]. We now report that Ca2+ inhibited IP3 binding to lbs-3 with an IC50 of approx. 700 nM (37±4% inhibition at 5 μM Ca2+), while IP3 binding to lbs-2 was not affected by increasing [Ca2+] from 100 nM to 25 μM. Calmodulin (10 μM) inhibited IP3 binding to lbs-3 by 37±4%, while IP3 binding to lbs-2 was inhibited by only 11±2%. The inhibition of IP3 binding to lbs-3 by calmodulin was dose-dependent (IC50≈ 2 μM). We conclude that the IP3-binding domains of the various IP3R isoforms differ in binding characteristics for IP3 and adenophostin A, and are differentially modulated by Ca2+ and calmodulin, suggesting that the various IP3R isoforms can have different intracellular functions.

Expression of mRNAs for different types of IP3 receptors in rat kidneys

1995 ◽  
Vol 268 (6) ◽  
pp. F1046-F1052 ◽  
Author(s):  
T. Yang ◽  
Y. Terada ◽  
H. Nonoguchi ◽  
K. Tomita ◽  
F. Marumo
Keyword(s):  
Collecting Duct ◽  
Rat Kidney ◽  
Ip3 Receptors ◽  
Ip3 Receptor ◽  
Receptor Mrna ◽  
Collecting Ducts ◽  
Different Types ◽  
Medullary Collecting Duct

Cloning studies have extensively characterized two types of inositol 1,4,5-trisphosphate (IP3) receptors from the rat. An IP3 receptor from the cerebellum is referred to as type 1, and a second, recently described, receptor is referred to as the type 2 IP3 receptor. The significance of different types of IP3 receptors, especially in vivo in the kidney, is not fully understood. We investigated the localization of mRNAs encoding these two types of IP3 receptors in microdissected nephron segments of rats using reverse transcription and polymerase chain reaction (RT-PCR) followed by Southern blot analysis. Type 1 IP3 receptor mRNA displayed a widespread, although not uniform, distribution along the nephron. In contrast, type 2 IP3 receptor mRNA was confined almost exclusively to collecting ducts, suggesting specific expression of type 2 IP3 receptor in collecting ducts. We then detected mRNAs for the two types of IP3 receptors in collecting ducts in dehydrated rats. Dehydration downregulated type 2 IP3 receptor mRNA in cortical collecting duct, outer medullary collecting duct, and the initial part of inner medullary collecting duct (IMCD), but not in the terminal part of IMCD. It had no effect on type 1 IP3 receptor mRNA expression in collecting ducts. We propose that different types of IP3 receptors may have different functions in the rat kidney. the initial part of inner medullary collecting duct (IMCD), but not in the terminal part of IMCD. It had no effect on type 1 IP3 receptor mRNA expression in collecting ducts. We propose that different types of IP3 receptors may have different functions in the rat kidney.

scholarly journals Stimulation of Inositol 1,4,5-Trisphosphate (IP3) Receptor Subtypes by Adenophostin A and Its Analogues

PLoS ONE ◽  
2013 ◽  
Vol 8 (2) ◽  
pp. e58027 ◽  
Author(s):  
Huma Saleem ◽  
Stephen C. Tovey ◽  
Andrew M. Riley ◽  
Barry V. L. Potter ◽  
Colin W. Taylor
Keyword(s):  
Receptor Subtypes ◽  
Ip3 Receptor ◽  
Inositol 1,4,5 Trisphosphate ◽  
Adenophostin A ◽  
Stimulation Of

scholarly journals Ca2+-calmodulin inhibits Ca2+ release mediated by type-1, -2 and -3 inositol trisphosphate receptors

2000 ◽  
Vol 345 (2) ◽  
pp. 357-363 ◽  
Author(s):  
Charles E. ADKINS ◽  
Stephen A. MORRIS ◽  
Humbert DE SMEDT ◽  
Ilse SIENAERT ◽  
Katalin TÖRÖK ◽  
...  
Keyword(s):  
Calcineurin Inhibitors ◽  
Cell Types ◽  
Receptor Subtypes ◽  
Glutathione S Transferase ◽  
Calmodulin Binding ◽  
Insp3 Receptors ◽  
Adenophostin A ◽  
A Site ◽  
Insp3 Receptor

InsP3 binding to type-1, but not type-3, InsP3 receptors is inhibited by calmodulin in a Ca2+-independent fashion [Cardy and Taylor (1998) Biochem. J. 334, 447-455], and Ca2+ mobilization by type-1 InsP3 receptors of cerebellum is inhibited by calmodulin [Patel, Morris, Adkins, O'Beirne and Taylor (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 11627-11632]. Using cell types expressing predominantly type-1, -2 or -3 InsP3 receptors, we show that InsP3-evoked Ca2+ mobilization from each is similarly inhibited by calmodulin. In SH-SY5Y cells, which express largely type-1 receptors, calmodulin (IC50 ≈ 15 μM) inhibited InsP3-evoked Ca2+ release only in the presence of Ca2+. The inhibition was unaffected by calcineurin inhibitors. The effect of calmodulin did not result from enhanced metabolism of InsP3 because calmodulin also decreased the sensitivity of the Ca2+ stores to adenophostin A, a non-metabolizable InsP3-receptor agonist. Protein kinase A-catalysed phosphorylation of type-1 InsP3 receptors was unaffected by Ca2+-calmodulin. Using a scintillation proximity assay to measure 125I-calmodulin binding to glutathione S-transferase-fusion proteins, we identified two regions of the type-1 InsP3 receptor (cyt1, residues -6 to 159; and cyt11, residues 1499-1649) that bound 125I-calmodulin. The higher-affinity site (cyt11) was also photoaffinity labelled with N-hydroxysuccinimidyl-4-azidobenzoate (HSAB)-calmodulin. We speculate that Ca2+-independent binding of calmodulin to a site within the first 159 residues of the type-1 InsP3 receptor inhibits InsP3 binding and may thereby regulate the kinetics of Ca2+ release. Ca2+-dependent inhibition of Ca2+ release by calmodulin is mediated by a different site: it may reside on an accessory protein that associates with all three receptor subtypes, or Ca2+-calmodulin binding to a site lying between residues 1499 and 1649 of the type-1 receptor may inhibit Ca2+ release from any tetrameric receptor that includes a type-1 subunit.

Limited Proteolysis of Vitronectin by Plasmin Destroys Heparin Binding Activity

1991 ◽  
Vol 66 (03) ◽  
pp. 310-314 ◽  
Author(s):  
David C Sane ◽  
Tammy L Moser ◽  
Charles S Greenberg
Keyword(s):  
Limited Proteolysis ◽  
Plasminogen Activator Inhibitor ◽  
Binding Activity ◽  
Binding Domain ◽  
Heparin Binding ◽  
Inhibitor Type ◽  
Activator Inhibitor Type ◽  
Activator Inhibitor ◽  
Pai 1

SummaryVitronectin (VN) stabilizes plasminogen activator inhibitor type 1 (PAI-1) activity and prevents the fibrin(ogen)-induced acceleration of plasminogen activation by t-PA. These antifibrinolytic activities as well as other functions are mediated by the glycosaminoglycan (GAG) binding domain of VN. Since the GAG binding region is rich in arginyl and lysyl residues, it is a potential target for enzymes such as plasmin. In this paper, the dose and time-dependent proteolysis of VN by plasmin is demonstrated. The addition of urokinase or streptokinase (200 units/ml) to plasma also produced proteolysis of VN. With minimal proteolysis, the 75 kDa band was degraded to a 62-65 kDa form of VN. This minimal proteolysis destroyed the binding of [3H]-heparin to VN and reversed the neutralization of heparin by VN.Thus, the plasmin-mediated proteolysis of the GAG binding activity of VN could destroy the antifibrinolytic activity of VN during physiologic conditions and during thrombolytic therapy. Furthermore, other functions of VN in complement and coagulation systems that are mediated by the GAG binding domain may be destroyed by plasmin proteolysis.

Sign in / Sign up

Export Citation Format

Share Document