Molecular target sizes of inositol 1,4,5-trisphosphate receptors in liver and cerebellum

Ins(1,4,5)P3 is the intracellular messenger that mediates the effects of many cell-surface receptors on intracellular Ca2+ stores. Although radioligand-binding studies have identified high-affinity Ins(1,4,5)P3-binding sites in many tissues, these have not yet been convincingly shown to be the receptors that mediate Ca2+ mobilization, nor is it clear whether there are differences in these binding sites between tissues. Here we report that Ins(1,4,5)P3 binds to a single class of high-affinity sites in both permeabilized hepatocytes (KD = 7.8 +/- 1.1 nM) and cerebellar membranes (KD = 6.5 +/- 2.4 nM), and provide evidence that these are unlikely to reflect binding to either of the enzymes known to metabolize Ins(1,4,5)P3. Furthermore, the rank order of potency of synthetic inositol phosphate analogues in displacing specifically bound Ins(1,4,5)P3 is the same as their rank order of potency in stimulating mobilization of intracellular Ca2+ stores, suggesting that the Ins(1,4,5)P3-binding site may be the physiological receptor. Radiation inactivation of the Ins(1,4,5)P3-binding sites of liver and cerebellum reveals that they have similar molecular target sizes: 257 +/- 36 kDa in liver and 258 +/- 20 kDa in cerebellum. We conclude that an Ins(1,4,5)P3-binding protein with a molecular target size of about 260 kDa is probably the receptor that mediates Ca2+ mobilization in hepatocytes, and our limited data provide no evidence to distinguish this from the cerebellar Ins(1,4,5)P3-binding protein.

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Human basophils express interleukin-4 receptors

Blood ◽

10.1182/blood.v76.9.1734.bloodjournal7691734 ◽

1990 ◽

Vol 76 (9) ◽

pp. 1734-1738

Author(s):

P Valent ◽

J Besemer ◽

K Kishi ◽

F Di Padova ◽

K Geissler ◽

...

Keyword(s):

Binding Sites ◽

Interleukin 4 ◽

Target Cells ◽

Specific Cell ◽

Binding Studies ◽

Single Class ◽

Chronic Granulocytic Leukemia ◽

High Affinity ◽

F Cells ◽

Human Basophils

Interleukin-4 (IL-4), a multipotential lymphokine reputed to play an important role in the regulation of immune responses, interacts with a variety of hemopoietic target cells through specific cell surface membrane receptors. The present study was designed to investigate whether human basophils express IL-4 binding sites. For this purpose, basophils were enriched to homogeneity (93% and 98% purity, respectively) from the peripheral blood of two chronic granulocytic leukemia (CGL) donors using a cocktail of monoclonal antibodies (MoAbs) and complement. Purified basophils bound 125I-radiolabeled recombinant human (rh) IL-4 in a specific manner. Quantitative binding studies and Scatchard plot analysis revealed the presence of a single class of high affinity IL-4 binding sites (280 +/- 40 sites per cell in donor 1 and 640 +/- 45 sites per cell in donor 2) with an apparent dissociation constant, kd, of 7.12 x 10(-11) +/- 2.29 x 10(-11) and 9.55 +/- 3.5 x 10(-11) mol/L, respectively. KU812-F, a human basophil precursor cell line, was found to express a single class of 810 to 1,500 high affinity IL-4 binding sites with a kd of 2.63 to 5.54 x 10(-10) mol/L. No change in the numbers or binding constants of IL-4 receptors was found after exposure of KU812-F cells to rhIL-3 (a potent activator of basophils) for 60 minutes. No effect of rhIL-4 on 3H-thymidine uptake, release or synthesis of histamine, or expression of basophil differentiation antigens (Bsp-1, CD11b, CD25, CD40, CD54) on primary human CGL basophils or KU812-F cells was observed.

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α1-Adrenoceptor subtypes on rat afferent arterioles assessed by radioligand binding and RT-PCR

AJP Renal Physiology ◽

10.1152/ajprenal.2001.281.1.f172 ◽

2001 ◽

Vol 281 (1) ◽

pp. F172-F178 ◽

Cited By ~ 16

Author(s):

Max Salomonsson ◽

Melinda Oker ◽

Susan Kim ◽

Hua Zhang ◽

James E. Faber ◽

...

Keyword(s):

Binding Sites ◽

Radioligand Binding ◽

Rt Pcr ◽

Binding Studies ◽

Single Class ◽

Site Model ◽

Adrenoceptor Antagonist ◽

Resistance Vessels ◽

Afferent Arterioles ◽

Radioligand Binding Studies

We utilized [3H]prazosin saturation and competition radioligand binding studies to characterize the expression of α1-adrenoceptors in preglomerular vessels. mRNA for adrenoceptor subtypes was assayed using RT-PCR. The vessels were isolated using an iron oxide-sieving method. [3H]prazosin bound to a single class of binding sites ( K d0.087 ± 0.012 nM, Bmax 326 ± 56 fmol/mg protein). Phentolamine displaced [3H]prazosin (0.2 nM) with a p K i of 8.37 ± 0.09. Competition with the selective α1A-adrenoceptor antagonist 5-methylurapidil fit a two-site model (p K i9.38 ± 0.21 and 7.04 ± 0.15); 59 ± 3% of the sites were high-affinity, and 41 ± 3% were low-affinity binding sites. Competition with the α1D-adrenoceptor antagonist 8-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-8-azaspiro[4.5]decane-7,9-dione dihydrochloride (BMY-7378) fit a one-site model with low affinity (p K i 6.83 ± 0.03). The relative contents of α1A-, α1B-, and α1D-adrenoceptor mRNAs were 64 ± 5, 25 ± 5, and 11 ± 1%, respectively. Thus there was a very good correlation between mRNA and receptor binding for the subtypes. These data indicate a predominance of the α1A-adrenoceptor subtype in rat renal resistance vessels, with smaller densities of α1B- and α1D-adrenoceptors.

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Characterization of beta-adrenergic receptors in cultured bovine tracheal gland cells

AJP Cell Physiology ◽

10.1152/ajpcell.1989.256.2.c310 ◽

1989 ◽

Vol 256 (2) ◽

pp. C310-C314 ◽

Cited By ~ 5

Author(s):

J. M. Madison ◽

C. B. Basbaum ◽

J. K. Brown ◽

W. E. Finkbeiner

Keyword(s):

Binding Sites ◽

Adrenergic Receptors ◽

Adrenergic Receptor ◽

Rank Order ◽

Beta Adrenergic Receptors ◽

Binding Studies ◽

Beta Adrenergic ◽

High Affinity ◽

Gland Cells ◽

Beta 2

We characterized the beta-adrenergic receptors that mediate secretory responses to isoproterenol in cultured bovine tracheal submucosal gland cells. Previous studies have shown that these cells have morphological and biochemical features characteristic of serous cells. Isoproterenol, epinephrine, and norepinephrine each stimulated the secretion of 35SO4-labeled macromolecules from these cultured serous cells with a rank order of potency (isoproterenol greater than epinephrine greater than norepinephrine) consistent with the presence of beta 2-adrenergic receptors. These functional studies were supported by radioligand-binding studies using [I125]-iodocyanopindolol (125I-CYP) to identify beta-adrenergic receptors. 125I-CYP binding to membrane particulates prepared from cultured serous cells was saturable and of high affinity (equilibrium dissociation constant 20 +/- 3 pM; mean +/- SE, n = 6) and was antagonized stereoselectively by propranolol. Adrenergic agonists competed for 125I-CYP-binding sites with a rank order of potency characteristic of the beta 2-adrenergic receptor subtype. A specific beta 2-adrenergic receptor antagonist, ICI 118.551, competed for a single class of 125I-CYP-binding sites with high affinity (inhibition constant 1.8 +/- 0.3 nM, n = 3). We concluded that the secretory response of cultured tracheal gland cells to isoproterenol is a response mediated by beta-adrenergic receptors of the beta 2 subtype.

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Identification and transmembrane signaling of leukotriene D4 receptors in human mesangial cells

AJP Cell Physiology ◽

10.1152/ajpcell.1988.255.6.c771 ◽

1988 ◽

Vol 255 (6) ◽

pp. C771-C780 ◽

Cited By ~ 31

Author(s):

M. S. Simonson ◽

P. Mene ◽

G. R. Dubyak ◽

M. J. Dunn

Keyword(s):

Binding Sites ◽

Radioligand Binding ◽

Mesangial Cells ◽

Specific Binding ◽

Transmembrane Signaling ◽

Binding Studies ◽

Single Class ◽

Leukotriene D4 ◽

Human Mesangial Cells ◽

Leukotriene Receptors

Although peptidoleukotriene (LTC4, LTD4) receptors have been characterized by radioligand binding studies, pathways of transmembrane signaling by activated leukotriene receptors remain obscure. We employed [3H]LTD4 binding studies and fluorescent measurements of intracellular Ca2+ concentration ([Ca2+]) and pH to identify LTD4 receptors and mechanisms of transmembrane signaling in cultured human mesangial cells. Mesangial cells expressed a single class of saturable, specific binding sites for [3H]LTD4. Kinetic, competition, and saturation analyses gave an average KD of approximately 12.0 nM with a Bmax of 987 fmol/mg protein. LTC4 competed with high affinity for [3H]LTD4 binding sites, as did LTB4 but with much lower affinity. [3H]LTD4 binding was blocked by a specific LTD4 receptor antagonist, SKF 102922. LTD4 and LTC4 also evoked a rapid (2-3 s), transient increase in intracellular [Ca2+], followed by a second, sustained increase. The transient phase was independent of extracellular Ca2+, whereas the sustained phase was dependent on extracellular Ca2+. Intracellular [Ca2+] was unaffected by LTB4. The LTD4-stimulated Ca2+ transients were dose dependent (1 nM-1 microM) and, similar to [3H]LTD4 binding, Ca2+ transients were inhibited by LTD4 receptor antagonists. We also report evidence that LTD4 affects intracellular pH and activates Na+-H+ exchange. Specifically, LTD4 induced an initial acidification within 1-2 min, followed by net alkalinization at 5 min. Alkalinization was due to activation of an amiloride-inhibitable Na+-H+ exchanger. LTD4 receptors were apparently not coupled to adenylate cyclase or phospholipase A2 as we detected no changes of adenosine 3',5'-cyclic monophosphate (cAMP) or prostanoids. Thus we conclude that [3H]LTD4 binding sites on human mesangial cells are coupled to a Ca2+-signaling system and Na+-H+ exchange. Moreover LTD4, a potent inflammatory mediator, failed to stimulate cAMP or prostaglandin E2/prostaglandin I2, two counterregulatory autacoids that preserve normal mesangial function.

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Enhanced responsiveness of endothelium in the growing/motile state to tumor necrosis factor/cachectin.

Journal of Experimental Medicine ◽

10.1084/jem.170.3.913 ◽

1989 ◽

Vol 170 (3) ◽

pp. 913-931 ◽

Cited By ~ 40

Author(s):

H Gerlach ◽

H Lieberman ◽

R Bach ◽

G Godman ◽

J Brett ◽

...

Keyword(s):

Endothelial Cell ◽

Binding Sites ◽

Cell Shape ◽

Radioligand Binding ◽

Cell Binding ◽

Binding Studies ◽

Endothelial Cell Surface ◽

High Affinity ◽

Radioligand Binding Studies

Some in vivo observations have suggested that growing or perturbed endothelium, such as that which occurs during angiogenesis, is more sensitive to the action of cytokines (TNF/cachectin, TNF, or IL-1) than normal quiescent endothelial cells. This led us to examine the responsiveness of endothelium to TNF as a function of the growth/motile state of the cell. TNF-induced modulation of endothelial cell surface coagulant function was half-maximal at a concentration of approximately 0.1 nM in subconfluent cultures, whereas 1-2 nM was required for the same effect in postconfluent cultures. Perturbation of endothelial cell shape/cytoskeleton was similarly more sensitive to TNF in subconfluent cultures. Consistent with these results, radioligand binding studies demonstrated high affinity TNF binding sites, Kd approximately 0.1 nM on subconfluent cultures, whereas only lower affinity sites (Kd approximately 1.8 nM) were detected on postconfluent cultures. The mechanisms underlying this change in the affinity of endothelium for TNF were studied in four settings. Crosslinking experiments with 125I-TNF and endothelium showed additional bands corresponding to Mr approximately 66,000 and approximately 84,000 with subconfluent cultures that were not observed with postconfluent cultures. Experiments with X-irradiated endothelium, whose growth but not motility was blocked, indicated that proliferation was not required for induction of high affinity TNF sites. Postconfluent endothelium, triggered to enter the proliferative cycle by microbutuble poisons, expressed high affinity TNF binding sites together with changes in cell shape/cytoskeleton well before their entry into S phase. Using wounded postconfluent monolayers, cells that migrated into the wound and those close to the wound edge displayed enhanced TNF binding and modulation of coagulant properties. These results suggest a model for targetting TNF action within the vasculature; regulation of high affinity endothelial cell binding sites can direct TNF to activated cells in particular parts of the vascular tree.

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A high-affinity oestrogen-binding protein in cockerel liver cytosol

Biochemical Journal ◽

10.1042/bj1800347 ◽

1979 ◽

Vol 180 (2) ◽

pp. 347-353 ◽

Cited By ~ 29

Author(s):

C B Lazier ◽

A J Haggarty

Keyword(s):

Binding Sites ◽

Proteinase Inhibitors ◽

Specific Binding ◽

Binding Protein ◽

Gel Filtration ◽

Binding Activity ◽

Single Class ◽

High Affinity ◽

Significant Concentration

In contrast with several earlier reports, cytosol from cockerel liver contains a significant concentration of a protein that binds oestradiol with high affinity. To demonstrate the activity, certain alterations in the conventional method of preparation of cytosol must be made. Homogenization in sucrose-containing buffer at pH 8.4 in the presence of proteinase inhibitors and rapid fractionation of the cytosol with (NH4)2SO4 enables demonstration of a single class of oestradiol-binding sites with a Kd of about 1 nM and specificity only for oestrogens. The concentration is about 300 sites per cell in liver from 2-week-old cockerels. Oestradiol treatment in vivo decreases the number of exchangeable cytosol oestradiol-binding sites by about 80% for 1–4h, after which time it is gradually restored. Gel filtration of the cytosol preparation in the presence of high salt concentrations reveals that most of the oestradiol-binding activity is in high-molecular-weight aggregates, but a mild trypsin treatment generates a specific binding protein with an approximate mol.wt. of 40 000. This protein may be an oestrogen receptor.

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Human basophils express interleukin-4 receptors

Blood ◽

10.1182/blood.v76.9.1734.1734 ◽

1990 ◽

Vol 76 (9) ◽

pp. 1734-1738 ◽

Cited By ~ 15

Author(s):

P Valent ◽

J Besemer ◽

K Kishi ◽

F Di Padova ◽

K Geissler ◽

...

Keyword(s):

Binding Sites ◽

Interleukin 4 ◽

Target Cells ◽

Specific Cell ◽

Binding Studies ◽

Single Class ◽

Chronic Granulocytic Leukemia ◽

High Affinity ◽

F Cells ◽

Human Basophils

Abstract Interleukin-4 (IL-4), a multipotential lymphokine reputed to play an important role in the regulation of immune responses, interacts with a variety of hemopoietic target cells through specific cell surface membrane receptors. The present study was designed to investigate whether human basophils express IL-4 binding sites. For this purpose, basophils were enriched to homogeneity (93% and 98% purity, respectively) from the peripheral blood of two chronic granulocytic leukemia (CGL) donors using a cocktail of monoclonal antibodies (MoAbs) and complement. Purified basophils bound 125I-radiolabeled recombinant human (rh) IL-4 in a specific manner. Quantitative binding studies and Scatchard plot analysis revealed the presence of a single class of high affinity IL-4 binding sites (280 +/- 40 sites per cell in donor 1 and 640 +/- 45 sites per cell in donor 2) with an apparent dissociation constant, kd, of 7.12 x 10(-11) +/- 2.29 x 10(-11) and 9.55 +/- 3.5 x 10(-11) mol/L, respectively. KU812-F, a human basophil precursor cell line, was found to express a single class of 810 to 1,500 high affinity IL-4 binding sites with a kd of 2.63 to 5.54 x 10(-10) mol/L. No change in the numbers or binding constants of IL-4 receptors was found after exposure of KU812-F cells to rhIL-3 (a potent activator of basophils) for 60 minutes. No effect of rhIL-4 on 3H-thymidine uptake, release or synthesis of histamine, or expression of basophil differentiation antigens (Bsp-1, CD11b, CD25, CD40, CD54) on primary human CGL basophils or KU812-F cells was observed.

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Specific endothelin binding sites in renal medullary collecting duct cells: lack of interaction with ANP binding and cGMP signalling

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y92-162 ◽

1992 ◽

Vol 70 (8) ◽

pp. 1167-1174 ◽

Cited By ~ 14

Author(s):

Peter Cernacek ◽

Louis Legault ◽

Duncan J. Stewart ◽

Mortimer Levy

Keyword(s):

Atrial Natriuretic Peptide ◽

Natriuretic Peptide ◽

Binding Sites ◽

Collecting Duct ◽

Specific Cell ◽

Binding Studies ◽

Single Class ◽

High Affinity ◽

Medullary Collecting Duct ◽

Atrial Natriuretic

The diverse biological actions of endothelins (ET) appear to be mediated by specific cell-surface receptors. Autoradiography and membrane binding studies have shown abundant ET binding sites in the kidney. However, their expression in specific types of renal cells is unclear. We studied the binding of 125I-labelled endothelin-1 in freshly isolated cell suspensions from canine inner medullary collecting duct. Competition binding experiments revealed the presence of specific high-affinity binding sites: unlabelled ET-1 and ET-2 competed with the radioligand with an IC50 of 135 and 83 pM, respectively, while the IC50 of ET-3 and big ET-1 were 2 and 4 orders of magnitude higher, indicating the presence of ETA-type receptor. Angiotensin II, vasopressin, and atrial natriuretic peptide (ANP) did not compete for ET binding even at a concentration of 10−6 M. Saturation binding experiments showed a single class of binding sites of high density (Bmax = 56.7 ± 10.3 fmol/106 cells) and high affinity (Kd = 69.8 ± 10 pM). In contrast, ANP receptors in the same cell preparations appeared as two classes of binding sites with widely different affinity and density. The high-affinity ANP site (Kd = 311 ± 48 pM) was compatible with ANP-B (guanylate cyclase-coupled) receptor. ET-1 did not compete for this receptor. ET-1 (10−7 M) did not alter ANP-induced cGMP generation in these cells (3.8-fold increase at 10−7 M ANP), nor basal levels of cGMP. The expression by the distal tubular epithelium of specific ET-1 binding sites strongly suggests the presence of a functional receptor, which may mediate the inhibition of Na+ transport in these cells. The mechanism and the transduction pathway of this effect appear to be different and independent from those of ANP.Key words: endothelin receptor, distal collecting duct, atrial natriuretic peptide receptor, cGMP generation.

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Direct measurement of vascular α1-adrenoceptors

Clinical Science ◽

10.1042/cs068s035 ◽

1985 ◽

Vol 68 (s10) ◽

pp. 35s-37s

Author(s):

Brian B. Hoffman ◽

Gozoh Tsujimoto

Keyword(s):

Direct Measurement ◽

Binding Sites ◽

Vascular Tone ◽

Radioligand Binding ◽

Rabbit Aorta ◽

Single Class ◽

Selective Antagonist ◽

High Affinity ◽

Adrenoceptor Regulation ◽

Α1 Receptors

1. α-Adrenoceptors play an important role in regulating vascular tone. [125I]BE2254, a high affinity antagonist, has been utilized to label α1-receptors in membrane preparations from rabbit aorta. [125I]BE2254 specifically labels a single class of binding sites with the characteristics of α1-receptors. 2. Catecholamines compete for [125I]BE2254 binding stereospecifically and with the characteristic α-adrenergic potency series (−)-adrenaline ≥ (−)-noradrenaline ≪ (−)-isoprenaline. 3. The α1-adrenoceptor selective antagonist prazosin is much more potent than yohimbine in competing for the [125I]BE2254 binding sites, which suggests that the α-adrenoceptor identified is predominantly of the α1 subtype. 4. The extension of radioligand binding techniques to individual rabbit aortas should simplify the study of vascular α-adrenoceptor regulation.

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Investigation of the forms of pig duodenal Ca2+-binding protein produced by limited tryptic hydrolysis

Biochemical Journal ◽

10.1042/bj2370781 ◽

1986 ◽

Vol 237 (3) ◽

pp. 781-787 ◽

Cited By ~ 1

Author(s):

D T W Bryant ◽

S Critch

Keyword(s):

Binding Site ◽

Binding Sites ◽

Binding Protein ◽

Native Protein ◽

Binding Studies ◽

High Affinity ◽

C Terminus ◽

Kd Values ◽

T1 And T2 ◽

Tryptic Hydrolysis

Vitamin D-dependent Ca2+-binding protein from pig duodenum was hydrolysed with trypsin in the presence of Ca2+ and two products were obtained: T1, which differed from the native protein by loss of Ac-Ser-Ala-Gln-Lys from the N-terminus and Ile-Ser-Gln-OH from the C-terminus, and T2, which differed from T1 by loss of a C-terminal lysine. The hydrolysis inactivated one of the two high-affinity Ca2+-binding sites on the native protein, and the remaining site was stable in T1 but labile in T2 when the proteins were Ca2+-free. Binding studies showed that T1 had Kd values of 2.8 +/- 0.1 nM, 57 +/- 13 microM and 0.8 +/- 0.3 microM for Ca2+, Mg2+ and Mn2+ respectively, and T2 had Kd 2.2 +/- 0.3 nM for Ca2+. The affinity for Mn2+, together with the other Kd values, identified the site on T1 as the site on the native protein previously found to have Kd 0.6 microM for Mn2+, rather than one with Kd 50 microM for Mn2+. In contrast with both the native protein and another form of the protein with a single Ca2+-binding site, the intrinsic fluorescence of T1 and T2 was little affected by the addition of Ca2+. It was concluded that the active binding site in T1 and T2, and also the site in the native protein with the higher affinity for Mn2+, was probably in the C-terminal half of the molecule.

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