The Mammalian β-Adrenergic Receptor: Structural and Functional Characterization of the Carbohydrate Moiety

Abstract

Mammalian β-adrenergic receptors are glycoproteins consisting of a single polypeptide chain of M_r ～64,000. Treatment of purified [¹²⁵I]-labeled hamster lung β-adrenergic receptor with α-mannosi-dase reveals two discrete populations of receptor consistent with previous studies using membrane bound photoaffinity-labeled receptor. Treatment of the [¹²⁵I]-labeled receptor with endo-glycosidase F results initially in the formation of a M_r ～57,000 peptide which is further converted to M_r ～49,000 suggesting that there are two N-linked carbohydrate chains per receptor polypeptide. Exoglycosidase treatments and lectin chromatography of the [¹²⁵I]-labeled receptor reveals the presence of two complex type carbohydrate chains (～10% of which are fucosylated) on ～45% of the receptors. The remaining ～55% of the receptors appear to contain a mixture of carbohydrate chains (possibly high mannose, hybrid and complex type chains). Deglycosylation of the receptor by endoglycosidase F does not appear to alter the binding affinity of the receptor for a variety of β-adrenergic agonists and antagonists. Moreover, the ability of control, α-mannosidase sensitive or insensitive (fractionated on immobilized wheat germ agglutinin) and neuraminidase, α-mannosidase or endoglycosidase F treated receptors to interact with the stimulatory guanine nucleo-tide regulatory protein in a reconstituted system were virtually identical. The deglycosylated receptor was also unaltered in its heat lability as well as its susceptibility to a variety of proteases. These findings demonstrate that the carbohydrate portion of the β-receptor does not contribute to determining either its specificity of ligand binding or coupling to the adenylate cyclase system.     

In Vivo Differences between Two Optical Isomers of Radioiodinated o-iodo-trans-decalinvesamicol for Use as a Radioligand for the Vesicular Acetylcholine Transporter

Purpose

To develop a superior VAChT imaging probe for SPECT, radiolabeled (-)-OIDV and (+)-OIDV were isolated and investigated for differences in their binding affinity and selectivity to VAChT, as well as their in vivo activities.

Procedures

Radioiodinated o-iodo-trans-decalinvesamicol ([¹²⁵I]OIDV) has a high binding affinity for vesicular acetylcholine transporter (VAChT) both in vitro and in vivo. Racemic [¹²⁵I]OIDV was separated into its two optical isomers (-)-[¹²⁵I]OIDV and (+)-[¹²⁵I]OIDV by HPLC. To investigate VAChT binding affinity (Ki) of two OIDV isomers, in vitro binding assays were performed. In vivo biodistribution study of each [¹²⁵I]OIDV isomer in blood, brain regions and major organs of rats was performed at 2,30 and 60 min post-injection. In vivo blocking study were performed to reveal the binding selectivity of two [¹²⁵I]OIDV isomers to VAChT in vivo. Ex vivo autoradiography were performed to reveal the regional brain distribution of two [¹²⁵I]OIDV isomers and (-)-[¹²³I]OIDV for SPECT at 60 min postinjection.

Results

VAChT binding affinity (Ki) of (-)-[¹²⁵I]OIDV and (+)-[¹²⁵I]OIDV was 22.1 nM and 79.0 nM, respectively. At 2 min post-injection, accumulation of (-)-[¹²⁵I]OIDV was the same as that of (+)-[¹²⁵I]OIDV. However, (+)-[¹²⁵I]OIDV clearance from the brain was faster than (-)-[¹²⁵I]OIDV. At 30 min post-injection, accumulation of (-)-[¹²⁵I]OIDV (0.62 ± 0.10%ID/g) was higher than (+)-[¹²⁵I]OIDV (0.46 ± 0.07%ID/g) in the cortex. Inhibition of OIDV binding showed that (-)-[¹²⁵I]OIDV was selectively accumulated in regions known to express VAChT in the rat brain, and ex vivo autoradiography further confirmed these results showing similar accumulation of (-)-[¹²⁵I]OIDV in these regions. Furthermore, (-)-[¹²³I]OIDV for SPECT showed the same regional brain distribution as (-)-[¹²⁵I]OIDV.

Conclusion

These results suggest that radioiodinated (-)-OIDV may be a potentially useful tool for studying presynaptic cholinergic neurons in the brain.     

Photoaffinity labeling the agonist binding domain of α4β4 and α4β2 neuronal nicotinic acetylcholine receptors with [I]epibatidine and 5[I]A-85380

The development of nicotinic acetylcholine receptor (nAChR) agonists, particularly those that discriminate between neuronal nAChR subtypes, holds promise as potential therapeutic agents for many neurological diseases and disorders. To this end, we photoaffinity labeled human α4β2 and rat α4β4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [¹²⁵I]epibatidine and 5[¹²⁵I]A-85380. Our results show that both agonists photoincorporated into the β4 subunit with little or no labeling of the β2 and α4 subunits respectively. [¹²⁵I]epibatidine labeling in the β4 subunit was mapped to two overlapping proteolytic fragments that begin at β4V102 and contain Loop E (β4I109-P120) of the agonist binding site. We were unable to identify labeled amino acid(s) in Loop E by protein sequencing, but we were able to demonstrate that β4Q117 in Loop E is the principal site of [¹²⁵I]epibatidine labeling. This was accomplished by substituting residues in the β2 subunit with the β4 homologs and finding [¹²⁵I]epibatidine labeling in β4 and β2F119Q subunits with little, if any, labeling in α4, β2, or β2S113R subunits. Finally, functional studies established that the β2F119/β4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation.     

Hepatic subcellular metabolism of heme from heme-hemopexin: incorporation of iron into ferritin

The subcellular distribution and metabolic fate of [⁵⁹Fe]heme-[¹²⁵I]-labeled hemopexin after receptor-mediated interaction with the liver was examined in the rat. After intravenous injection, [⁵⁹Fe]heme from the complex and ⁵⁹Fe from hepatic catabolism of this heme accumulate in the liver and undergo changes in their subcellular distribution over 2 hours. The amounts of [⁵⁹Fe]heme and particularly of ⁵⁹Fe increase in the cytosol while remaining constant or decreasing in membranous fractions. In contrast, [¹²⁵I]-labeled hemopexin associated with the liver during heme transport is always a small fraction of the dose and is not measurably catabolized under these conditions.Gel filtration of the cytosol showed that ⁵⁹Fe increased linearly with time in a high molecular weight fraction which was identified immunologically as ferritin. We conclude that heme transported by hemopexin is metabolized by the liver and the iron conserved.     

Conversion and binding of tetraiodothyronine in developing rat brain

In this study we have examined whether rat brain nuclear thyroid hormone receptors bind T₄ or metabolites of T₄ and whether there is a developmental change in brain T₄ metabolism and binding. Developing animals were injected with trace [¹²⁵I]3,5-tetraiodothyronine ([¹²⁵I]T₄) and after sacrifice brain nuclear and cytoplasmic fractions were examined to determine whether their radioactivity was represented by the injected [¹²⁵I]T₄ or any of its metabolites. Of the radiothyronines specifically bound to the nucleus, 90% was found to be triiodothyronine ([¹²⁵I] T₃) and 10% was [¹²⁵I]T₄. Of the cytoplasmic, protamine sulfate-precipitable fraction, 40% was [¹²⁵I]T₄ and 60% [¹²⁵I]T₃. Inasmuch as the percentage of [¹²⁵I] T₃ found in plasma during the same postinjection interval was similar to that present as contaminant of the injected material, it was concluded that brain [¹²⁵I] T₃ derives from local monodeiodination of T₄ to T₃. The main developmental change observed was a marked decline in the total cytoplasmic and nuclear [¹²⁵I] T₄ uptake. However, with development, the T₃/T₄ ratio remained constant in the nuclear fraction while it decreased in the cytoplasmic fraction. It is concluded that although T₃, deriving from monodeiodianation of T₄, is the main form of thyroid hormone that regulates brain development by its binding to brain nuclear receptors, the fact that T₄ is the most available from during the critical period makes it, indirectly, very important to brain development. Further, the decline observed with development in T₄ uptake and monodeiodination to T₃, may contribute to the concomitantly declining role of thyroid hormones on brain tissue.     

Synthesis and in vitro evaluation of radioiodinated indolequinones targeting NAD(P)H: Quinone oxidoreductase 1 for internal radiation therapy

NAD(P)H: quinone oxidoreductase 1 (NQO1) is an obligate two-electron reductase and is highly expressed in many human solid cancers. Because NQO1 can be induced immediately after exposure to ionizing radiation, we aimed to develop an NQO1-targeted radiolabeled agent to establish a novel internal radiation therapy that amplifies the therapeutic effects when combined with external radiation therapy. We designed three NQO1-targeted radioiodinated compounds including two ether linkage compounds ([¹²⁵I]1 and [¹²⁵I]2) and a sulfide linkage compound ([¹²⁵I]3) based on the selective binding of indolequinone analogs to the active site of NQO1 by the stacking effect. These compounds were successfully prepared using an oxidative iododestannylation reaction with high radiochemical yields and purity. In NQO1-expressing tumor cells, [¹²⁵I]1 and [¹²⁵I]2 were readily metabolized to p-[¹²⁵I]iodophenol or m-[¹²⁵I]iodophenol and [¹²⁵I]I⁻, whereas over 85% of the initial radioactivity of [¹²⁵I]3 was observed as an intact form at 1 h after incubation. The cellular uptake of [¹²⁵I]3 was significantly higher than those of [¹²⁵I]1 and [¹²⁵I]2. The uptake of [¹²⁵I]3 was specific and was dependent on the expression of NQO1. These data suggest that the novel NQO1-targeted radioiodinated compound [¹²⁵I]3 could be used as a novel internal radiation agent for the treatment of cancer.     

Identification of Surface Antigens of Sarcocystis muris (Coccidia)

Zoites of Sarcocystis muris were recovered from the skeletal muscles of infected mice by trypsin digestion. Extracts of zoites prepared by freeze-thaw, Triton X-100 (0.1%), or a combination of the two treatments contained antigenic components. Testing of these antigens by agar gel diffusion and immunoelectrophoresis against sera from infected mice showed one major precipitin band. SDS-polyacrylamide-gel electrophoresis (SDS-PAGE) of the extracts revealed at least eight detectable polypeptides ranging in molecular weight from 10,000 to 220,000. The antigenic components of the extract were identified by labeling the parasite surface with [¹²⁵I] and precipitation of the [¹²⁵I]-labeled antigens with immune sera. Analysis of the immunoprecipitates by SDS-PAGE and autoradiography revealed three antigens with molecular weights of 27,500, 43,000 and 90,000. The smallest of these was the predominant antigen as suggested by labeling intensity.     

The electrolytic preparation of bioactive radioiodinated parathyroid hormone of high specific activity.

A technique for the preparation of microgram quantities of bovine parathyroid hormone (bPTH) labeled with carrier-free ¹²⁵I to a specific activity of 1300 Ci/mmol is described. A restructured and simplified apparatus was used for electrolytic iodination, making it feasible to use reaction volumes of 100 to 200 ml. The miniaturized setup requires only a small platinum crucible connected via an agar-KCl salt bridge to a saturated KCl solution, a battery to drive the reaction, and a voltmeter to monitor the potential difference between the reference-saturated KCl solution (via a calomel electrode) and the platinum crucible. The [¹²⁵I]-labeled bPTH elutes as a single species when chromatographed on a Biogel P-10 column equilibrated in 3 m guanidine HCl-2.3 m formic acid, and it retains full biologic activity when bioassayed in vivo. It is evident that bPTH labeled to a high specific activity with ¹²⁵I does not suffer in regard to its biological potency.     

Assessment of [125I]WYE-230949 as a Novel Histamine H3 Receptor Radiopharmaceutical

Histamine H₃ receptor therapeutics have been proposed for several diseases such as schizophrenia, attention deficit hyperactivity disorder, Alzheimer''s disease and obesity. We set out to evaluate the novel compound, [¹²⁵I]WYE-230949, as a potential radionuclide imaging agent for the histamine H₃ receptor in brain. [¹²⁵I]WYE-230949 had a high in vitro affinity for the rat histamine H₃ receptor (K_d of 6.9 nM). The regional distribution of [¹²⁵I]WYE-230949 binding sites in rat brain, demonstrated by in vitro autoradiography, was consistent with the known distribution of the histamine H₃ receptor. Rat brain uptake of intravenously injected [¹²⁵I]WYE-230949 was low (0.11 %ID/g) and the ratio of specific: non-specific binding was less than 1.4, as determined by ex vivo autoradiography. In plasma, metabolism of [¹²⁵I]WYE-230949 into a less lipophilic species occurred, such that less than 38% of the parent compound remained 30 minutes after injection. Brain uptake and metabolism of [¹²⁵I]WYE-230949 were increased and specific binding was reduced in anaesthetised compared to conscious rats. [¹²⁵I]WYE230949 is not a potential radiotracer for imaging rat histamine H₃ receptors in vivo due to low brain uptake, in vivo metabolism of the parent compound and low specific binding.     

Disposition of125I-labeled recombinant human tumor necrosis factor in the tumor-bearing mouse

Disposition of [¹²⁵I]rHu-TNF was elucidated in BALB/c mice bearing Meth A fibrosarcoma 7 days after transplantation. Afteri.v. administration, [¹²⁵I]rHu-TNF measured by radioactivity and immunoreactivity biphasically decreased in plasma. Tumor level of [¹²⁵I]rHu-TNF was the maximum at 1 h, then decreased and finally remained essentially constant. After i.t. administration, plasma level reached the maximum at 1 h. Tumor level decreased quickly and then became essentially constant. [¹²⁵I]rHu-TNF was suggested to be degraded to small fragments in the tumor. Significant distribution of [¹²⁵I]rHu-TNF was found in the kidney, lung, liver and tumor. Most tissue levels decreased with time in parallel with plasma levels. [¹²⁵I]rHu-TNF radioactivity was found in proximal convoluted tubules of kidney and in those areas of tumor consisting of degenerating cells with pyknotic nuclei. Urine contained most of administered radioactivity, which being neither immunoreactive nor protein-bound.     

Identification of a high-affinity binding protein for N-acetylchitooligosaccharide elicitor in the plasma membrane of suspension-cultured rice cells by affinity labeling

A high-affinity binding protein for the N-acetylchito-oligosaccharide elicitor of phytoalexin biosynthesis was identified by photoaffinity labeling and affinity cross-linking in the plasma membrane of suspension-cultured rice cells. Both a [¹²⁵I]-labeled photolabile 2-(4-azidophenyl)ethylamino conjugate ([¹²⁵I]-GN₈-AzPEA) and a [¹²⁵I]-labeled 2- (4-aminophenyl)ethylamino conjugate ([¹²⁵]-GN₈-APEA) of N-acetylchito-octaose were synthesized. The two conjugates were separately incubated with the plasma membrane prepared by aqueous two-phase partitioning, and covalently cross-linked to the elicitor binding site by irradiation with UV light or treatment with the cross-linking agent glutaraldehyde, respectively. Autoradiography of the SDS-PAGE gel of the solubilized membrane proteins revealed the labeling of a single 75 kDa band in both cases. The incorporation of the radiolabeled ligands into the 75 kDa protein showed a saturable mode of binding, with half-maximal incorporation at 45 and 52 nM for photoaffinity labeling and affinity cross-linking, respectively. The labeling of the 75 kDa protein was inhibited by N-acetylchito-oligasaccharides in a size-dependent manner, and N-acetylchito-octaose (GlcNAc)₈ showed a half-maximal inhibition at concentrations of the order of 10 nM. However, neither chito-octaose (GlcN)₈, cellopentaose nor α-1,4 linked N-acetylgalactosamine octamer (GalNAc)₈ at concentrations as high as 25 μM inhibited the labeling of the 75 kDa protein. These results are in good agreement with the sensitivity and the specificity of the ‘high-affinity binding site’ previously identified by binding assays, as well as with the activities of these oligosaccharides in the induction of phytoalexin biosynthesis and other cellular responses. These results suggest that the 75 kDa protein identified by the affinity labeling represents a functional receptor for this elicitor.     

The use of homologous and hetebologous 125 I-radioligands in the radioimmunoassay of progesterone

Eight homologous and heterologous¹²⁵ I-radioligand systems for the radioimmunoassay of progesterone were examined. Using an antiserum raised to 11α-hydroxyprogesterone 11-succinyl-bovine serum albumin, standard curves were set up with the homologous radioligands, 11α-hydroxyprogesterone 11-succinyl-[¹²⁵I]-iodotyramine, -[¹²⁵I]-iodohistamine and -[¹²⁵I]-iodotyrosine methyl ester. Heterologous bridge systems were represented by progesterone-11α-oxycarbonyl-[¹²⁵I]-iodotyrosine methyl ester and 11α-hydroxyprogesterone 11-phthalyl-[¹²⁵I]-iodotyrosine methyl ester, and heterologous site systems by progesterone-3-(O-carboxymethyl)oxime-[¹²⁵I]-iodotyramine, progesterone-12-(O-carboxymethyl) oxime-[¹²⁵ I]-iodotyramine, and progesterone-20-(O-carboxymethyl) oxime-[¹²⁵I]-iodohistamine. The preparation of the steroid derivatives and iodination by a two-phase method are described. The curves obtained from the homologous radioligands were relatively insensitive compared with a tritiated system, with the tyrosine methyl ester derivative providing a more sensitive assay than the corresponding tyramine or histamine analogues. The heterologous bridge systems gave more sensitive curves than the homologous tracers whilst the 3- and 12-(O-carboxymethyl) oxime derivatives of progesterone furnished curves as sensitive as the tritiated reference. Progesterone-20-(O-carboxymethyl)oxime-[¹²⁵I]-iodohistamine was not bound by the antibody.     

3-(Trifluoromethyl)-3-(m-isothiocyanophenyl)diazirine: Synthesis and chemical characterization of a heterobifunctional carbene-generating crosslinking reagent

A new hydrophobic heterobifunctional photocrosslinking reagent 3-(trifluoromethyl)-3-(m-isothiocyanophenyl)diazirine (TRIMID), a carbene precursor, and its radioiodinated analogue [¹²⁵I]TRIMID, have been synthesized and chemically characterized. The reagents were applied for membrane protein modification in human erythrocyte membranes and purple membranes fromHalobacterium halobium. Covalent labeling of the anion transport protein (band 3) via the isothiocyanate function was confirmed. Radiolabeled TRIMID was detected in at least two thermolysin-generated transmembrane fragments of the anion transport protein, and half-maximal inhibition of the erythrocyte anion transport activity was attained with 2.2 mM reagent. In bacteriorhodopsin (BR), a common binding site for the monofunctional phenylisothiocyanate and the bifunctional crosslinking reagent was identified: preincubation of purple membranes with TRIMID suppressed phenylisothio-[¹⁴C]-cyanate binding to BR. [¹²⁵I]TRIMID was recovered in V-1, the N-terminal segment of BR, which includes the phenylisothiocyanate binding site Lys-41. Light-induced intramolecular crosslinking of band 3-derived thermolytic fragments was not observed, although the carbene was generatedin situ and photocrosslinking of the protease V8 fragments of BR was not detected. Chemical and physicochemical characteristics of the new reagent are discussed with regard to limitations imposed for photoinduced site-directed crosslink formation.     

Production of monoclonal antibodies against a cell surface concanavalin a binding glycoprotein

Concanavalin A-binding (Con A)-binding cell surface glycoproteins were isolated, via Con A-affinity chromatography, from Triton X-100-solubilized Chinese hamster ovary (CHO) cell plasma membranes. The Con A binding glycoproteins isolated in this manner displayed a significantly different profile on sodium dodecyl sulfate-polyacrylamide gels than did the Tritonsoluble surface components, which were not retarded by the Con A-Sepharose column. [¹²⁵I]-Con A overlays of the pooled column fractions displayed on sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) demonstrated that there were virtually no Con A receptors associated with the unretarded peak released by the Con A-Sepharose column, whereas the material which was bound and specifically eluted from the Con A-Sepharose column with the sugar hapten α-methyl-D-mannopyranoside contained at least 15 prominent bands which bound [¹²⁵I]-Con A. In order to produce monoclonal antibodies against various cell surface Con A receptors, Balb/c mice were immunized with the pooled Con A receptor fraction. Following immunization spleens were excised from the animals and single spleen cell suspensions were fused with mouse myeloma P3/X63-Ag8 cells. Numerous hybridoma clones were subsequently picked on the basis of their ability to secrete antibody which could bind to both live and glutaraldehyde-fixed CHO cells as well as to the Triton-soluble fraction isolated from the CHO plasma membrane fraction. Antibody from two of these clones was able to precipitate a single [¹²⁵I]-labeled CHO surface component of ～265,000 daltons.     

LOCALIZATION OF POLYSOME-BOUND ALBUMIN AND SERINE DEHYDRATASE IN RAT LIVER CELL FRACTIONS

The polysomes involved in albumin and serine dehydratase synthesis were identified and localized by the binding to rat liver polysomes of anti-rat serum albumin and anti-serine dehydratase [¹²⁵I]Fab dimer and monomer. Techniques were developed for the isolation of undegraded free and membrane-bound polysomes and for the preparation of [¹²⁵I]Fab monomers and dimers from the IgG obtained from the antisera to the two proteins, rat serum albumin and serine dehydratase. The distribution of anti-rat serum albumin [¹²⁵I]Fab dimer in the polysome profile is in accordance with the size of polysomes that are expected to be synthesizing albumin. By direct precipitation, it has been demonstrated that nascent chains isolated from the membrane-bound polysomes by puromycin were precipitated by anti-rat serum albumin-IgG at a level of 5–6 times those released from free polysomes. Anti-rat serum albumin-[¹²⁵I]Fab dimer reacted with membrane-bound polysomes almost exclusively compared to the binding of nonimmune, control [¹²⁵I]Fab dimer; a significant degree of binding of anti-rat serum albumin-[¹²⁵I]Fab to free polysomes was also obtained. The [¹²⁵I]Fab dimer made from normal control rabbit serum does not react with polysomes from liver at all and this preparation will not interact with polysomes extracted from tissues that do not synthesize rat serum albumin. Both anti-serine dehydratase-[¹²⁵I]Fab monomer and dimer react with free and bound polysomes from livers of animals fed a chow diet or those fed a high 90% protein diet and given glucagon. In the latter instance, however, it is clear that the majority of the binding occurs to the bound polysomes. Furthermore, the specificity of this reaction may be further shown by the use of kidney polysomes that do not normally synthesize serine dehydratase. When these latter polysomes are isolated, even after the addition of crude and purified serine dehydratase, no reaction with anti-serine dehydratase-Fab fragments could be demonstrated. These results indicate that the reaction of the Fab fragments are specific for polysomes that synthesize rat serum albumin or rat liver serine dehydratase. Furthermore, they demonstrate that even with this high degree of specificity, some polysomes in the fraction labeled "free" are in the process of synthesizing rat serum albumin while bound polysomes to a significant, if not major, degree are the site of the synthesis of rat liver serine dehydratase.     

Two Saturable Recognition Sites for (-)[125I]Iodo-N6-(4-Hydroxyphenyl-Isopropyl)-Adenosine Binding on Purified Cardiac Sarcolemma

Abstract

Analysis of (-)[¹²⁵]iodo-N⁶-(4-hydroxyphenylisopropyl)-adenosine ([¹²⁵I]HPIA) binding to purified sarcolemmal preparations of guinea pig and bovine hearts revealed two classes of binding sites when unlabeled iodo-HPIA (100 μmol/1) was used as non-specific binding marker. In the presence of 1 mmol/1 theophylline, however, only the high affinity component was detected. Adenosine receptor agonists caused biphasic displacement of [¹²⁵I]HPIA binding, with a high affinity potency rank order typical of interaction with A₁-adenosine receptors. Biphasic competition curves were also observed with 8-phenyltheophylline and isobutylmethylxanthine, whereas the theophylline curve was monophasic up to 1 mmol/1. In brain membranes, specific binding of [¹²⁵I]HPIA as well as of [³H]PIA was further reduced when unlabeled iodo-HPIA replaces theophylline as the non-specific binding marker. These results suggest the presence of two [¹²⁵I]HPIA binding sites on cardiac sarcolemma and brain membranes, but receptor function can only be ascribed to the high affinity sites. The low affinity site probably represents an artefact, which is often observed when non-specific binding is defined with the unlabeled counterpart or a structurally related ligand of the radioligand used.     

Labeling proteins using aryl iodide acylation agents: Influence of meta vs para substitution on in vivo stability

The effect of para vs meta substitution on the biological behavior of an intact antibody and an F(ab′)₂ fragment was investigated. Paired-label studies were performed using 81C6 IgG and OC 125 F(ab′)₂ labeled using the N-succinimidyl esters of both p-[¹²⁵I]- and m-[¹³¹I]iodobenzoate as well as with the potential catabolites, p-[¹²⁵I]- and m-[¹³¹I]iodobenzoic acid. In all 3 studies, up to 55% lower uptake of ¹³¹I in thyroid and stomach was observed, suggesting that the m-substituted species were more inert to dehalogenation in vivo.     

A Study of the Cerebral Cortex Cholecystokinin Receptor Using Two Radiolabelled Probes: Evidence for a Common CCK 8 and CCK 4 Cholecystokinin Receptor Binding Site

Abstract: This study was directed at the issue of whether or not subpopulations of cholecystokinin (CCK) receptors exist within the CNS. This was achieved through the use of two radiolabelled probes, namely [¹²⁵I] Bolton-Hunter (BH) CCK 8 and [³H]pentagastrin (Boc-β-Ala CCK 4), in comparative studies under identical conditions. Both probes bound with high affinity to the mouse cerebral cortical CCK receptor binding site with apparent equilibrium dissociation constants (K_D) of 1.9 nM and 1.4 nM for [³H]pentagastrin and [¹²⁵I]BH CCK 8, respectively. The maximal binding capacity was 1.05 and 1.15 pmol/g weight for the tritium and iodinated probes, respectively. Hill analysis yielded Hill numbers close to unity, suggesting the absence of more than one binding site and the lack of cooperativity of CCK receptor binding. Kinetic studies revealed binding site homogeneity in that no evidence of multiphasic dissociation curves was seen. Computerised analysis of displacement binding data using LIGAND established that both radiolabelled probes bound to a single site, with the one-site model providing the best fit of the data. Similar rank orders of potency were obtained for various fragments of CCK 8 in competing for the CCK receptor, labelled with either probe. Both CCK 8 and CCK 4 bound with roughly equinanomolar affinity. These studies demonstrate that both CCK 8 and its shorter C-terminal fragment CCK 4 bind to a single class of high-affinity binding site, with as yet no evidence of CNS CCK receptor multiplicity.     

The Elusive Nature of Cerebellar Somatostatin Receptors: Studies in Rat,Monkey and Human Cerebellum

Abstract

The pharmacological profile and localization of somatostatin (SRIF) receptors were determined in rat, monkey and human cerebellum. In rat cerebellar cortex, low ss₁/sst₄, intermediate sst₂ and very high sst₃ receptor mRNA levels were found, sst₁ mRNA was also expressed in the deep cerebellar nuclei. [¹²⁵I]Tyr³-octreotide binding sites in cerebellar membranes correlated with recombinant sst₂, but not with sst₅ or sst₃ receptors and were found in the molecular layer of the cerebellum. [¹²⁵I]CGP 23996 (in Na⁺-buffer) binding in rat cerebellum correlated with sst₁ or sst₄, but not with sst₂, sst₃ or sst₅ receptor binding. Similar data were obtained in rhesus monkey cerebellum. mRNAs for all five receptors were found in the granule cell layer of the human cerebellum and/or in the dentate nucleus. [¹²⁵I]Tyr³-octreotide binding was strong in the molecular layer and correlated with that of recombinant sst₂ receptors, but not with sst₃ or sst₅ receptors. [¹²⁵I]CGP 23996 (in Mg⁺⁺-buffer) binding was heterogeneous (about 75%. to sst₂ and 25% to sst₁ and/or sst₄ receptors). The molecular and granular layers were equally and the dentate nucleus strongly labeled. Thus. SRIF receptors of the sst₂, sst₁ and/or sst₄ subtype are present in the rat, monkey and human cerebellum. In the latter two species, the sst₂ type appears to be predominant. Surprisingly, the high expression of sst₃ receptor mRNA is not supported by radioligand binding data in any of the species studied. The reason for this discrepancy remains to be elucidated.     

Localization of cocaine analog [125I]RTI 82 irreversible binding to transmembrane domain 6 of the dopamine transporter

The site of cocaine binding on the dopamine transporter (DAT) was investigated using the photoactivatable irreversible cocaine analog [125I]3beta-(p-chlorophenyl)tropane-2beta-carboxylic acid, 4'-azido-3'-iodophenylethyl ester ([125I]RTI 82). The incorporation site of this compound was mapped to transmembrane domains (TMs) 4-6 using epitope-specific immunoprecipitation of trypsin fragments and further localized using cyanogen bromide (CNBr), which hydrolyzes proteins on the C-terminal side of methionine residues. CNBr hydrolysis of [125I]RTI 82-labeled rat striatal and expressed human DATs produced fragments of approximately 5-10 kDa consistent with labeling between Met(271/272) or Met(290) in TM5 to Met(370/371) in TM7. To further define the incorporation site, substitution mutations were made that removed endogenous methionines and inserted exogenous methionines in combinations that would generate labeled CNBr fragments of distinct masses depending on the labeling site. The results obtained were consistent with the presence of TM6 but not TMs 4, 5, or 7 in the labeled fragments, with additional support for these conclusions obtained by epitope-specific immunoprecipitation and secondary digestion of CNBr fragments with endoproteinase Lys-C. The final localization of [125I]RTI 82 incorporation to rat DAT Met(290)-Lys(336) and human DAT I291M to R344M provides positive evidence for the proximity of cocaine binding to TM6. Residues in and near DAT TM6 regulate transport and transport-dependent conformational states, and TM6 forms part of the substrate permeation pathway in the homologous Aquifex aeolicus leucine transporter. Cocaine binding near TM6 may thus overlap the dopamine translocation pathway and function to inhibit TM6 structural rearrangements necessary for transport.