Identification of somatostatin receptors by covalent labeling with a novel photoreactive somatostatin analog

We have synthesized two photoreactive derivatives of somatostatin, namely [125I-Tyr11,azidonitrobenzoyl (ANB)-Lys4]somatostatin and [125I-Tyr11,ANB-Lys9]somatostatin, and used them to characterize somatostatin receptors biochemically in several cell types. Saturation binding experiments carried out in the dark demonstrated that [125I-Tyr11,ANB-Lys4]somatostatin bound with high affinity (KD = 126 +/- 39 pM) to a single class of binding sites in GH4C1 pituitary cell membranes. The affinity of this analog was similar to that of the unsubstituted peptide [125I-Tyr11]somatostatin (207 +/- 3 pM). In contrast, specific binding was not observed with [125I-Tyr11,ANB-Lys9]somatostatin. The binding of both [125I-Tyr11,ANB-Lys4]somatostatin and [125I-Tyr11]somatostatin was potently inhibited by somatostatin (EC50 = 300 pM) whereas at 100 nM unrelated peptides had no effect. Furthermore, both pertussis toxin treatment and guanyl-5'yl imidophosphate (Gpp(NH)p) markedly reduced [125I-Tyr11,ANB-Lys4]somatostatin binding. Thus, [125I-Tyr11,ANB-Lys4]somatostatin binds to G-protein coupled somatostatin receptors with high affinity. To characterize these receptors biochemically, GH4C1 cell membranes were irradiated with ultraviolet light following the binding incubation, and the labeled proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. A major band of 85 kDa was specifically labeled with [125I-Tyr11,ANB-Lys4]somatostatin but not with [125I-Tyr11,ANB-Lys9]somatostatin or [125I-Tyr11]somatostatin. The binding affinity of the 85-kDa protein for [125I-Tyr11,ANB-Lys4]somatostatin was very high (Kd = 34 pM). Labeling of this protein was inhibited competitively by somatostatin (EC50 = 140 +/- 80 pM) but not by unrelated peptides. Furthermore, this band was not labeled in pertussis toxin-treated membranes or in untreated membranes incubated with Gpp(NH)p. Finally, [125I-Tyr11,ANB-Lys4]somatostatin specifically labeled bands of 82, 75, and 72 kDa in membranes prepared from mouse pituitary AtT-20 cells, rat pancreatic acinar AR4-2J cells, and HIT hamster islet cells, respectively. Thus, [125I-Tyr11,ANB-Lys4]somatostatin represents the first photolabile somatostatin analog able to bind to receptors with high affinity. Our studies demonstrate that this novel peptide covalently labels specific somatostatin receptors in a variety of target cell types.     

A reversible radioligand specific for the ETB receptor: [125I]Tyr13-Suc-[Glu9,Ala11,15]-endothelin-1(8- 21), [125I]IRL 1620.

Suc-[Glu9,Ala11,15]-endothelin(ET)-1(8-21), IRL 1620, is a linear ET-analog specific for the ET-isopeptide-nonselective ETB receptor. The radio-iodinated analog, [125I]IRL 1620, showed a single class of saturable binding to the ETB receptors in porcine lung membranes with a Kd of 18 pM and a Bmax of 930 fmol/mg protein, which are almost comparable to the values obtained with [125I]ET-3 (6 pM and 900 fmol/mg protein). In competitive binding assays with [125I]IRL 1620, unlabeled ET-1, ET-3, IRL 1620 and [monoiodo-Tyr13]-IRL 1620 showed almost identical displacement curves with Ki of 8 to 16 pM. However, [125I]IRL 1620 was dissociated from the binding sites by addition of an excess amount (100 nM) of any of these unlabeled peptides, each with the same t1/2 of 100 min. This was in marked contrast to [125I]ET-3 which was hardly dissociated from the binding sites.     

Hypothalamic binding sites for pituitary adenylate cyclase activating polypeptide: characterization and molecular identification.

The goal of these experiments was to identify and characterize binding sites in the rat hypothalamus for the peptide, pituitary adenylate cyclase activating polypeptide (PACAP). The 27 amino acid form of PACAP (PACAP27) was used as the radiolabeled ligand in these experiments. Binding of [125I]PACAP27 to hypothalamic membrane preparations was rapid, reversible on addition of unlabeled peptide, and at least partially regulated by GTP. Nonhydrolyzable GTP analogs, guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S), guanosine-5'-(2-thiodiphosphate) (GDP beta S), and guanylylimidophosphate (GppNHp) also displaced [125I]PACAP27 binding to hypothalamic membrane preparations in a dose-dependent manner. The order of potency for the three analogs was GTP gamma S greater than GDP beta S greater than GppNHp. Both forms of the peptide, PACAP27 and PACAP38, were highly potent in displacing bound [125I]PACAP27, whereas VIP or PACAP(1-23) were unable to displace binding at concentrations of up to 500 nM. Scatchard analysis of the PACAP27 and PACAP38 displacement curves revealed that the fit of both curves was consistent with a single class of high-affinity binding sites, although the site exhibited a greater affinity for PACAP38 compared with PACAP27 (PACAP27 Kd = 1452 +/- 59 pM; PACAP38 Kd = 175 +/- 13 pM; Bmax 23.2 +/- 1.1 pmol/mg protein). The possibility of the existence of a class of binding sites with extremely low affinity cannot be discounted. After covalent cross-linking of [125I]PACAP27 with its receptor, the molecular weights of the complexes were estimated by electrophoresis and autoradiography. A major band of 60 Kd was evident when membranes were incubated with VIP or PACAP(1-23). Previous incubation with unlabeled PACAP27 or PACAP38 eliminated visualization of this band. These results suggest that a specific, high-affinity binding site for PACAP27 is present in rat hypothalamus, and that this site shows a greater affinity for PACAP38 compared with PACAP27. The molecular weight of the peptide-receptor complex is 60,000 kDa, and therefore the receptor itself has an apparent molecular weight 57,000.     

Characterization of specific receptors for atrial natriuretic factor in bovine adrenal zona glomerulosa

We have recently shown that synthetic rat atrial natriuretic factor (ANF) directly inhibits mineralocorticoid and glucocorticoid secretion in cultured bovine adrenal cells with a potency of 100 pM. [125I]iodo-ANF was used in the present study to characterize potential receptor sites in bovine zona glomerulosa membranes. ANF binds to a class of high affinity binding sites with a pK of 10.2 and a density of 1.3 pmol/mg protein. Detailed competition curves with ANF document a class of high affinity sites with a pK of 10.2 and also a second class of lower affinity sites with a pK of 8.5. Nonspecific binding amounts to less than 10% of [125I]iodo-ANF binding at concentrations less than 100 pM. High affinity binding of [125I]iodo-ANF is reversible with a half-time of association of 15 minutes at 25 pM and a half-time of dissociation of 140 minutes. Monovalent cations Na, Li and K equipotently enhance [125I]iodo-ANF specific binding. Divalent cations Mg, Ca and Mn also increase [125I]iodo-ANF specific binding, with Mn being the most active cation. No effect of guanine nucleotide could be detected on ANF binding. The binding of [125I]iodo-ANF is very specific and is not inhibited by 1 microM angiotensin II, ACTH, VIP, somatostatin, Leu-enkephalin, dynorphin or by the N-terminal of POMC. The N-terminal fragment ANF-(1-16) is also completely inactive. Reduction of the disulfide bridge of ANF inactivates the peptide. This enabled the development of a highly specific radio-receptor assay for ANF with a minimum detectable dose of 2 femtomoles. The results document the specific receptor involved in the potent inhibitory effect of ANF on adrenal steroidogenesis and indicate that bovine adrenal zonal glomerulosa provide a highly sensitive system for studying the recently discovered atrial natriuretic factor.     

Characterization of picomolar affinity binding sites for [125I]-human calcitonin gene-related peptide in rat brain and heart

We have characterized picomolar affinity binding sites for human calcitonin gene-related peptide (CGRP) in rat brain and heart (atria and ventricle) membranes. By saturation analysis, apparent dissociation constant (KD) values of high affinity sites for [125I]-human CGRP are 9 approximately 15 pM (brain), 34 pM (ventricle) and 85 pM (atria). Low affinity sites with KD values of about 50 nM are found in rat brain and ventricle, but not in atria. Human and rat CGRP potently inhibited [125I]-human CGRP binding to these high affinity sites with apparent inhibition constant (Ki) values comparable to their KD values. Salmon calcitonin marginally inhibited these binding with Ki values between 0.1 microM and 1 microM. Extremely potent cardiovascular and gastrointestinal actions of CGRP might be mediated through CGRP binding sites with picomolar affinity which are similar to those we characterized in this study.     

Antibodies Against Synthetic Peptides Predicted from the Nucleotide Sequence of D2 Receptor Recognize Native Dopamine Receptor Protein in Rat Striatum

Two peptides corresponding to amino acid sequences predicted from the nucleotide sequence of the dopamine D2 receptor were synthesized. Peptide I (CGSEG-KADRPHYC) and peptide II (NNTDQNECIIY), corresponding to 24-34 and 176-185 from the NH2 terminus, respectively, were conjugated to keyhold limpet hemocyanin and injected into rabbits. Peptide I showed a greater immunogenic response than did peptide II. Both peptide antibodies exhibited high titer for the homologous antigens, but showed little or no cross-reactivity with heterogeneous peptides. Peptide I antibodies reacted with striatal membrane proteins of apparent molecular masses of 120, 90, 85, and 30 kDa on a western blot. Furthermore, the 90-kDa band was identified as denatured D2 receptor by its high affinity for the D2 selective photoaffinity probe 125I-N'-azidospiperone (125I-NAPS). Photoaffinity labeling of the 90-kDa protein by 125I-NAPS was reduced by 40% in the presence of the peptide I antibody. In addition, evidence is also presented to show the low level of 90-kDa protein in cerebellum which contains little or no D2 ligand binding sites. The antibody to peptide I inhibited the binding of [3H]YM-09151-2, a dopamine D2 receptor selective antagonist, to striatal membranes in a concentration-dependent manner; a 50% inhibition was obtained at a 1:500 dilution of the antisera with 20 pM ligand concentration. The data on the equilibrium inhibition kinetics of [3H]YM-09151-2 binding to striatal membranes were examined in the presence of antibody and showed a 25-30% decrease in Bmax (203.5 +/- 11.0 and 164.6 +/- 3.3 fmol/mg of protein in presence of preimmune and immune sera, respectively) with no change in KD.(ABSTRACT TRUNCATED AT 250 WORDS)     

Guanine Nucleotides Regulate 2-[125I]Iodomelatonin Binding Sites in Chick Retinal Pigment Epithelium but Not in Neuronal Retina

The characteristics of the binding sites labeled by the radioligand 2-[125I]iodomelatonin were compared in chicken neuronal retina and retinal pigment epithelium (RPE). Specific binding of 2-[125I]iodomelatonin in both sites was stable, saturable, reversible, and of high affinity. Scatchard analysis revealed an affinity constant (KD) of 446 +/- 55 pM and a total number of binding sites (Bmax) of 25.4 +/- 2.2 fmol/mg of protein for neuronal retina. For RPE the KD was 34.1 +/- 2.2 pM and the Bmax 59.5 +/- 5.2 fmol/mg of protein. Competition experiments with various melatonin analogues gave the following order of affinities: 2-iodomelatonin greater than 2-chloromelatonin greater than melatonin greater than 6-chloromelatonin greater than 6-hydroxymelatonin greater than N-acetylserotonin greater than 6-methoxyharmalan greater than 5-hydroxytryptamine. Linear regression of log Ki values from neuronal retina and RPE gave a highly significant correlation (r = 0.994, n = 8; p less than 0.001). GTP inhibited specific binding to RPE membranes in a concentration-dependent manner, but not in neuronal retinal membranes. The present results strongly suggest that a single type of melatonin receptor is found in neuronal retina and RPE, and that the site in RPE is coupled to a guanine nucleotide-binding regulatory protein (G protein), but that in neuronal retina is not.     

Atrial natriuretic factor (ANF) binding sites in frog kidney and adrenal.

Atrial natriuretic factor (ANF) binding sites were localized and quantified in kidney and adrenal of the frog Rana temporaria by quantitative in vitro autoradiography. [125I]-rat ANF(99-126) binding was present in kidney glomeruli and in the outer layer of interrenal tissue in the adrenal gland. ANF binding exhibited positive cooperativity with a half-maximal binding concentration (EC50) of 102 +/- 16 pM in glomeruli and 93 +/- 19 pM in interrenal tissue (n = 8). The corresponding maximal binding capacities (Bmax) were 1.33 +/- 0.16 and 1.21 +/- 0.36 fmol/mm2. [125I]-Rat ANF(99-126) binding was competitively displaced by unlabeled ANF analogues with an intact disulfide bridge showing a lower affinity than the iodinated ligand. The presence of ANF binding in glomeruli and steroidogenic interrenal cells suggests physiological functions of ANF for the osmomineral regulation in the frog by influencing glomerular filtration rate and adrenal steroid secretion.     

Differential effect of pH on thromboxane A2/prostaglandin H2 receptor agonist and antagonist binding in human platelets.

The effects of changes in pH on the binding of agonists and antagonists to the human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor were determined. Competition binding studies were performed with the TXA2/PGH2 mimetic [1S-1 alpha,2 beta (5Z), 3 alpha(1E,3R*),4 alpha)]-7-[3-(3-hydroxy-4'-iodophenoxy)-1-buteny) 7-oxabicyclo-[2.2.1]-heptan-2-yl]-5-heptenoic acid ([125I]BOP). The pH optimum for binding of [125I] BOP to washed human platelets was broad with a range of pH 4-6 in contrast to that of the TXA2/PGH2 receptor antagonist 9,11-dimethyl-methano-11,12-methano-16-(3-iodo-4-hydroxyl)-13-aza-15 alpha,beta-omega-tetranorthromboxane A2 ([125I]PTA-OH) which was 7.4. Scatchard analysis of [125I]BOP binding in washed platelets at pH 7.4, 6.0, and 5.0 revealed an increase in affinity (Kd = 1.16 +/- 0.06, 0.64 +/- 0.09, and 0.48 +/- 0.05 nM, respectively) and an increase in the number of receptors (Bmax = 2807 +/- 415, 5397 +/- 636, and 7265 +/- 753 sites/platelet, respectively). The potency of I-BOP to induce shape change in washed platelets at pH 6.0 was also significantly increased from an EC50 value of 0.34 +/- 0.016 nM at pH 7.4 to 0.174 +/- 0.014 nM at pH 6.0 (n = 6, p less than 0.05). In contrast, the EC50 value for thrombin was unaffected by the change in pH. In competition binding studies with [125I]BOP, the affinity of the agonists U46619 and ONO11113 were increased at pH 6.0 compared to 7.4. In contrast, the affinity of the TXA2/PGH2 receptor antagonists I-PTA-OH, SQ29548, and L657925 were either decreased or unchanged at pH 6.0 compared to 7.4. Diethyl pyrocarbonate and N-bromosuccinimide, reagents used to modify histidine residues, reversed the increase in affinity of [125I]BOP at pH 6.0 to values equivalent to those at pH 7.4. In solubilized platelet membranes, the effects of NBS were blocked by coincubation with the TXA2/PGH2 mimetic U46619. The results suggest that agonist and antagonist binding characteristics are different for the TXA2/PGH2 receptor and that histidine residue(s) may play an important role in the binding of TXA2/PGH2 ligands to the receptor.     

Angiotensin II binding sites in frog kidney and adrenal.

Angiotensin II (AII) binding sites were localized and quantified in kidney and adrenal of the frog Rana temporaria by quantitative in vitro autoradiography. AII binding was present in kidney glomeruli and in interrenal tissue of the outer zone of the adrenal gland. Saturation experiments showed that [125I]-[Val5]AII binds to a single class of binding sites with a dissociation constant (Kd) of 548 +/- 125 pM in glomeruli and 593 +/- 185 pM in interrenal tissue (n = 8). The corresponding maximal binding capacities (Bmax) were 2.48 +/- 0.71 and 3.05 +/- 1.02 fmol/mm2, respectively. AII binding was displaced by unlabeled angiotensin analogues in the rank order: [Sar1]AII greater than human AII greater than [125I]-[Val5]AII = [Val5]AII = human AIII much greater than human AI. The AII binding sites in glomeruli and interrenal tissue suggest an influence of AII on glomerular filtration rate and adrenal steroid secretion to take part in osmomineral regulation of the frog.     

Characterization of Binding Sites for the Angiotensin II Antagonist 125I-[Sarc1,Ile8]-Angiotensin II on Murine Neuroblastoma N1E-115 Cells

The murine neuroblastoma N1E-115 cell line contains binding sites for the angiotensin II (Ang II) receptor antagonist 125I-[Sarc1,Ile8]-Ang II (125I-SARILE). Binding of 125I-SARILE to N1E-115 membranes was rapid, reversible, and specific for Ang II-related peptides. The rank order potency of 125I-SARILE binding was the following: [Sarc1]-Ang II = [Sarc1,Ile8]-Ang II greater than Ang II greater than Ang III = [Sarc1,Thr8]-Ang II much greater than Ang I. Scatchard analysis of membranes prepared from confluent monolayers revealed a homogenous population of high affinity (KD = 383 +/- 60 pM) binding sites with a Bmax of 25.4 +/- 1.6 fmol/mg of protein. Moreover, the density, but not the affinity, of the binding sites increased as the cells progressed from logarithmic to stationary growth in culture. Finally, agonist, but not antagonist, binding to N1E-115 cells was regulated by guanine nucleotides. Collectively, these results suggest that the murine neuroblastoma N1E-115 cell line may provide a useful model in which to investigate the signal transduction mechanisms utilized by neuronal Ang II receptors.     

Characterization of functional calcitonin gene-related peptide receptors on rat lymphocytes.

Calcitonin gene-related peptide (CGRP), a vasoactive neuropeptide present in peripheral neurons, is released at local sites of inflammation. In these studies specific high affinity adenylyl cyclase linked CGRP receptors were characterized on rat lymphocytes. The distribution, affinity, and specificity of CGRP receptors was analyzed by radioligand binding. 125I-[His10]CGRP binding to rat lymphocytes was rapid, reaching equilibrium by 20 to 30 min at 22 degrees C, and dependent on cell concentration. The dissociation constants, Kd, for the CGRP receptor on purified T and B lymphocytes are 0.807 +/- 0.168 nM and 0.387 +/- 0.072 nM and the densities are 774 +/- 387 and 747 +/- 244 binding sites/cell, respectively. Competition binding studies determined that rat CGRP inhibits 125I-[His10]CGRP binding to lymphocytes with the highest affinity (Ki = 0.192 +/- 0.073) followed by human CGRP and the CGRP receptor antagonist CGRP8-37. 125I-[His10]CGRP binding to rat lymphocytes was not inhibited by the neuropeptides substance P, calcitonin, or neuropeptide Y. Lymphocyte CGRP receptor proteins were identified by affinity labeling by using disuccinimidyl suberate to covalently cross-link 125I-[His10]CGRP to its receptor. Specifically labeled CGRP binding proteins visualized by SDS-PAGE analysis had molecular masses of 74.5 and 220 kDa. A third high molecular mass protein band which did not penetrate the gel was also observed. In functional studies, CGRP stimulated a rapid, sustained increase in cAMP with an ED50 of approximately 8 pM. In experiments comparing optimal concentrations of isoproterenol, a beta 2-adrenergic agonist, and CGRP, intracellular cAMP elevation after isoproterenol treatment returned to basal levels by 30 min, whereas cAMP was still elevated at 60 min after CGRP treatment. The response to CGRP was specific in that it could be completely blocked by CGRP8-37. The presence of high affinity functional CGRP receptors on T and B lymphocytes provides evidence for a modulatory role for CGRP in regulating lymphocyte function.     

A novel radioligand [125I]BQ-3020 selective for endothelin (ETB) receptors.

A linear endothelin (ET) analog, N-acetyl-LeuMetAspLysGluAlaValTyrPheAlaHisLeu-AspIleIleTrp (BQ-3020), is highly selective for ETB receptors. BQ-3020 displaces [125I]ET-1 binding to ETB receptors (nonselective to ET isopeptides) in porcine cerebellar membranes (IC50: 0.2nM) at a concentration 4,700 times lower than that to ETA receptors (selective to ET-1) on aortic vascular smooth muscle cells (VSMC) (IC50: 940nM). BQ-3020 as well as ET-1 and ET-3 elicits vasoconstriction in the rabbit pulmonary artery. The ETA antagonist BQ-123 failed to inhibit this BQ-3020-induced vasoconstriction. Furthermore, BQ-3020 elicits endothelium-dependent vasodilation. These data indicate that BQ-3020 has ETB agonistic activity. The radioligand [125I]BQ-3020 binds to cerebellar membranes at single high affinity sites (Kd = 34.4pM), whereas it scarcely binds to VSMC. [125I]BQ-3020 binding to the cerebellum was displaced by BQ-3020, ET-1 and ET-3 in a nonselective manner (IC50: 0.07-0.17nM). However, the binding of [125I]BQ-3020 was insensitive to the ETA antagonist BQ-123 and other bioactive peptides. Both [125I]ET-1 and [125I]BQ-3020 show slow onset and offset binding kinetics to ETB receptors. These data indicate that the radioligand [125I]BQ-3020 selectively labels ETB receptors and that the slow binding kinetics of ET-1 are dependent on the peptide sequence from Leu6 to Trp21, but not on the structure formed by its two disulfide bridges.     

4-Iodotomoxetine: a novel ligand for serotonin uptake sites.

The tomoxetine analog, R-4-iodotomoxetine, binds in vitro to a single site of rat cortical membranes with high affinity (Kd = 0.03 +/- 0.01 nM, n = 4) and can be blocked by a selective serotonin reuptake site inhibitor, paroxetine. The [125I]R-4-iodotomoxetine binding at equilibrium is saturable and is temperature- and Na(+)-dependent. The number of specific [125I]R-4-iodotomoxetine binding sites (Bmax = 356 +/- 20 fmol/mg protein) is similar to that of [3H]citalopram (329 +/- 30 fmol/mg protein), a known serotonin uptake inhibitor. The binding of [125I]R-4-iodotomoxetine is selectively inhibited by several serotonin uptake blockers, and a good correlation is demonstrated between the potency of various drugs to inhibit in vitro binding of [125I]R-4-iodotomoxetine and [3H]citalopram. In addition, lesions performed with the neurotoxin p-chloroamphetamine, which destroys monoamine neurons, including serotonergic neuronal system, result in a 90% reduction of [125I]R-4-iodotomoxetine binding when compared to sham controls. These results indicate that the binding sites labeled by [125I]R-4-iodotomoxetine are associated with the neuronal serotonin uptake sites. However, the in vivo and ex vivo results do not show regional localization corresponding to the distribution of serotonin uptake sites. The nonspecific uptake may be related to this compound's high lipophilicity (octanol-buffer partition coefficient = 1100 - 1400 at pH 7). Although the in vivo properties of [125I]R-4-iodotomoxetine make it an unlikely candidate for mapping serotonin uptake sites with SPECT, the high affinity and selectivity should make it a useful tool for in vitro studies of the serotonin uptake sites.     

2-(125I) iodomelatonin binding sites in rat adrenals: pharmacological characteristics and subcellular distribution.

Specific binding sites for 2-[125I] iodomelatonin, a selective radiolabeled melatonin receptor ligand, were detected and characterized in rat adrenal membranes. Saturation studies demonstrated that 2-[125I]iodomelatonin binds to a single class of sites with an affinity constant (Kd) of 541 pM and a total binding capacity (Bmax) of 3.23 fmol/mg protein. Competition experiments revealed that the relative order of potency of compounds tested was as follows: 6-chloromelatonin greater than 2-iodomelatonin greater than melatonin greater than 5-methoxytryptamine greater than 5-methoxytryptophol. The highest density of binding sites was found in membranes from nuclear (0.76 fmol/mg protein) and mitochondrial (1.82 fmol/mg protein) subcellular fractions.     

Isoelectric and mass characterization of human platelet thromboxane A2/prostaglandin H2 receptors

To further characterize the human thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) receptor, preparative isoelectric focusing (IEF) was performed on solubilized platelet membranes. TXA2/PGH2 receptors, assayed by specific binding of the TXA2/PGH2 antagonist [125I]PTA-OH, were electrofocused at pH 5.6. Scatchard analysis of IEF fraction pH 5.6 revealed a 180-fold concentration of TXA2/PGH2 receptors (Bmax = 3650 +/- 228 pM/mg focused, 19 +/- 4 pM/mg unfocused) with no change in binding affinity (Kd = 47 +/- 7 nM focused, 36 +/- 14 nM unfocused). SDS-polyacrylamide gel electrophoresis of photoaffinity-labelled electrofocused receptors revealed concentration of specifically labelled proteins having molecular masses of 49,000 and 27,000 Daltons. These results suggest that the human platelet TXA2/PGH2 receptor has a pI of 5.6, molecular mass of 49,000 Daltons, and may exist as a dimer. Preparative IEF should prove useful in the eventual purification of this receptor.     

Covalent affinity labeling, detergent solubilization, and fluid-phase characterization of the rabbit neutrophil formyl peptide chemotaxis receptor

The formyl peptide chemotaxis receptor of rabbit neutrophils and purified rabbit neutrophil plasma membranes has been identified by several affinity labeling techniques: covalent affinity cross-linking of N-formyl-Nle-Leu-Phe-Nle-125I-Tyr-Lys (125I-hexapeptide) to the membrane-bound receptor with either dimethyl suberimidate or ethylene glycol bis(succinimidyl succinate) and photoactivation of N-formyl-Nle-Leu-Phe-Nle-125I-Tyr-N epsilon-[6-[(4-azido-2-nitrophenyl)amino]hexanoyl]Lys(125I-PAL). These techniques specifically identify the receptor as a polypeptide that migrates as a broad band on sodium dodecyl sulfate-polyacrylamide electrophoresis, with Mr 50 000-65 000. The receptor has been solubilized in active form from rabbit neutrophil membranes with the detergents 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and digitonin and from whole cells with CHAPS. Chemotaxis receptor activity was measured by the ability of the solubilized membrane material to bind 125I-hexapeptide or fMet-Leu-[3H]Phe with gel filtration or rapid filtration through poly(ethylenimine)- (PEI) treated filters as assay systems. 125I-PAL was specifically cross-linked to the same molecular weight material in the CHAPS and digitonin solubilized extract, but no specific labeling of the receptor was seen when membranes were extracted with Nonidet P-40 and Triton X-100. Therefore, although a large number of detergents are able to solubilize the receptor, it appears that some release the receptor in an inactive form. The ligand binding characteristics of fMet-Leu-[3H]Phe to the CHAPS-solubilized receptor shared properties with the membrane-bound formyl peptide receptor, both of which showed curvilinear, concave-upward Scatchard plots. Computer curve fitting with NONLIN and statistical analyses of the binding data indicated that for both the membrane-bound and solubilized receptors a two saturable sites model fitted the data significantly better (p less than 0.01) than did a one saturable site model. The characteristics of the two saturable sites model for the soluble receptor were a high-affinity site with a KD value of 1.25 +/- 0.45 nM and a low-affinity site with a KD value of 19.77 +/- 3.28 nM. A total of 35% of the two sites detected was of the higher affinity. In addition, a Hill coefficient of 0.61 +/- 0.12 was observed.     

Photoaffinity labeling of dopamine D1 receptors

A high-affinity radioiodinated D1 receptor photoaffinity probe, (+/-)-7-[125I]iodo-8-hydroxy-3-methyl-1-(4-azidophenyl)-2,3,4,5-tetra hyd ro- 1H-3-benzazepine ([125I]IMAB), has been synthesized and characterized. In the absence of light, [125I]IMAB bound in a saturable and reversible manner to sites in canine brain striatal membranes with high affinity (KD approximately equal to 220 pM). The binding of [125I]IMAB was stereoselectively and competitively inhibited by dopaminergic agonists and antagonists with an appropriate pharmacological specificity for D1 receptors. The ligand binding subunit of the dopamine D1 receptor was visualized by autoradiography following photoaffinity labeling with [125I]IMAB and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Upon photolysis, [125I]IMAB incorporated into a protein of apparent agents in a stereoselective manner with a potency order typical of dopamine D1 receptors. In addition, smaller subunits of apparent Mr 62,000 and 51,000 were also specifically labeled by [125I]IMAB in these species. Photoaffinity labeling in the absence or presence of multiple protease inhibitors did not alter the migration pattern of [125I]IMAB-labeled subunits upon denaturing electrophoresis in both the absence or presence of urea or thiol reducing/oxidizing reagents. [125I]IMAB should prove to be a useful tool for the subsequent molecular characterization of the D1 receptor from various sources and under differing pathophysiological states.     

Photoaffinity labeling of platelet activating factor binding sites in rabbit platelet membranes

A photoreactive, radioiodinated derivative of platelet activating factor (PAF), 1-O-(4-azido-2-hydroxy-3-iodobenzamido)undecyl-2-O-acetyl-sn- glycero-3-phosphocholine ([125I]AAGP), was synthesized and used as a photoaffinity probe to study the PAF binding sites in rabbit platelet membranes. The nonradioactive analog, IAAGP, induced rabbit platelet aggregation with an EC50 value of 3.2 +/- 1.9 nM as compared to 0.40 +/- 0.25 nM for PAF. Specific binding of [125I]AAGP to rabbit platelet membranes was saturable with a dissociation constant (Kd) of 2.4 +/- 0.7 nM and a receptor density (Bmax) of 1.1 +/- 0.2 pmol/mg protein. Photoaffinity labeling of platelet membranes with [125I]AAGP revealed several 125I-labeled components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A protein species with apparent molecular weight of 52,000 was consistently observed and inhibited significantly by unlabeled PAF at nanomolar concentrations. The labeling was specific since the PAF antagonists, SRI-63,675 and L-652,731, at 1 uM also blocked the appearance of this band; whereas lysoPAF was not effective at the same concentration. These results suggest that the binding sites of PAF receptor in rabbit platelets reside in the polypeptide of Mr = 52,000.     

Regulation of 1,4-dihydropyridine and beta-adrenergic receptor sites in coronary artery smooth muscle membranes.

The receptor sites for 1,4-dihydropyridine (DHP) calcium channel ligands were identified and pharmacologically characterized in partially purified canine coronary artery smooth muscle (CSM) membranes (purification factor for 1,4-DHPs 2.8 and 2.2 respectively) using Ca2+ channel agonist (-)-S-[3H]BAYK 8644 and antagonist (+)-[3H]PN 200-110 as radioligands. The beta-adrenergic receptors were identified with (-)-3-[125I]iodocyanopindolol (ICYP). Specific binding of 1,4-DHPs and ICYP to membrane fraction was saturable, reversible and of both high and low affinity. The Kd for 1,4-DHP Ca2+ channel agonist was 0.59 +/- 0.05 and for antagonist 0.35 +/- 0.06 nmol/l and for low affinity binding sites Kd = 9.0 +/- 0.18 and 18.0 +/- 1.1 nmol/l. The high affinity 1,4-DHP binding (Bmax = 265 +/- 21 and 492 +/- 12 fmol/mg protein), showed stereoselectivity, temperature-dependence as well as pharmacological specificity: isoprenaline- and GTP-sensitivity, positive modulation with dilthiazem and negative modulation with verapamil, that is, properties characteristic of 1,4-DHP receptor sites on L-type Ca2+ channels. The low affinity binding sites were characterized as nonselective, temperature independent, dipyridamol-sensitive and represented a nucleoside transporter. The proportion of high affinity binding sites identified in the CSM membranes was 1.85 : 1.0 in favour of the antagonist. Results obtained with [125I]omega Conotoxin GVI A demonstrated that CSM membrane fractions isolated from median layers of coronary artery were devoid of substantial contamination with fragments of neuronal cells.