Preparation of an iodinated radioligand.

Radioactive tracers are useful for receptor measurement and for radioimmunoassays because of the ease with which radioactive ligands are detected. Typical receptor measurement methods include whole tissue receptor assays, autoradiography, and cell and membrane binding assays. The isotopes most commonly used in receptor measurement studies are tritium, carbon-14, and iodine-125, with the choice of isotope most often based on the specific activity required and the sensitivity and type of detection system available. Its high specific activity and easy detection make I-125 ideal for labeling large peptides and proteins. While the short half-life (60 days) of iodine requires frequent replacement with freshly iodinated compounds, this is also an advantage, as unused portions of radioligand can be held for decay and disposed of as nonradioactive waste. This unit describes the iodination of proteins or peptides using the 125I-labeled Bolton Hunter reagent, a convenient, easy-to-use, nonoxidizing labeling reagent.

Neuropharmacological assessment of potential dopamine D4 receptor-selective radioligands.

Radiolabeled dopamine D4 receptor-selective agents ([3H]1-benzyl-4-[ N-(3-isopropoxy-2-pyridinyl)-N-methyl]-aminopiperidine maleate; [3 H]PNU-101958. and [125I]1-[4-iodobenzyl]-4-[ N-(3-isopropoxy-2-pyridinyl)-N-methyl]-aminopiperidine; [125I]RBI-257) were prepared and characterized. With D4.2- and D2L receptor-transfected cell membranes, [3H]PNU-101958 showed high dopamine D4 receptor affinity and selectivity, and potent inhibition by dopamine D4 receptor-selective compounds. However, its binding with rat brain homogenates showed little regional selectivity, and pharmacology inconsistent with selective dopamine D4 receptor labeling. Autoradiography indicated partial displacement of [3H]PNU-101958 by unlabeled dopamine D4 receptor ligands without regional selectivity, and lack of selective labeling with [125I]RBI-257. The results encourage further efforts to develop better dopamine D4 receptor-selective radioligands.

125I-Tyro-sauvagine: a novel high affinity radioligand for the pharmacological and biochemical study of human corticotropin-releasing factor 2 alpha receptors.

Corticotropin-releasing factor (CRF) receptors encoded by two distinct genes have recently been identified and termed CRF1 and CRF2. CRF and the non-mammalian-related peptide sauvagine bind to and activate CRF1 receptors with high affinity and equal potency. Although CRF is significantly weaker at the CRF2 receptor, sauvagine retains its high affinity interactions with this receptor subtype. We expressed the human CRF1 and CRF2 receptor subtypes in stable cell lines and characterized 125I-Tyr0-sauvagine, a high affinity radiolabel suitable for the pharmacological and functional profiles of these proteins. 125I-Tyr0-sauvagine has high affinity (200-400 PM) for CRF1 receptors and demonstrates a pharmacological profile identical to that of 125I-Tyr0-ovine CRF-labeled CRF1 receptors. 125I-Tyr0-sauvagine binding to human CRF2 alpha receptors is saturable and of high affinity (KD = 100-300 PM) and demonstrates guanine nucleotide sensitivity typical of agonist binding to receptors. The pharmacological profile of 125I-Tyr0-sauvagine binding to CRF2 alpha receptors with respect to inhibition by CRF-related analogs is similar to the agonist profile of potencies obtained by measurements of cAMP production stimulated by these analogs in CRF2 alpha expressing cell lines and distinct from the profile of the CRF1 receptor subtype. Thus, the related nonmammalian peptides sauvagine and urotensin have high affinity and rat/ human CRF and ovine CRF have lower affinity for CRF2 receptors labeled with 125I-Tyr0-sauvagine. Because the distribution of CRF1 and CRF2 alpha receptors has been demonstrated to be distinct, suggesting selective functional roles for each receptor subtype, the ability to label CRF2 alpha receptors with 125I-Tyr0-sauvagine in vitro represents a unique opportunity for the discovery of subtype-selective nonpeptide ligands, which would presumably target different aspects of CRF-mediated disorders. We have thus identified and characterized a novel high affinity radioligand for the labeling of CRF2 receptors.

A carboxyl-terminally truncated mutant and nonglycosylated A2a adenosine receptors retain ligand binding.

The amino acids that comprise the ligand binding sites of adenosine receptors have not been identified. Adenosine and its agonist analogues differ from ligands for the well studied biogenic amine receptors and rhodopsin in that the adenosine receptor agonists are larger, contain a ribose moiety, and are uncharged at physiological pH. Thus, the locations of the ligand binding pockets of the adenosine receptors could differ significantly from those of the biogenic amine receptors. This report describes the characterization of a purification-amenable truncated mutant of the canine A2a adenosine receptor and demonstrates that neither the long carboxyl-terminal tail nor the glycosidic moiety appears to be required for ligand binding. The dog thyroid A2a adenosine receptor cDNA (RDC8) was subcloned into the mammalian expression vector pCMV4. A mutant A2a construct, in which six histidines replaced residues 310-412 as the carboxyl terminus of the protein, also was prepared. When overexpressed transiently in COS M6 cells, the wild-type and mutant A2a receptors exhibited similar 2-[p-(2-[3H]carboxyethyl)phenylethylamino]-5'-N- ethylcarboxamidoadenosine saturation binding and competition curve profiles. The following biochemical techniques confirmed that the COS M6 cells were transcribing and translating A2a receptors of the expected molecular masses: (a) immunoblotting with an antipeptide antibody directed against the putative carboxyl-terminal side of the second extracellular loop (Tyr155-Val172) of the canine A2a adenosine receptor, (b) photoaffinity labeling with the A2a-selective agonist 125I-2-[4-[2-[2-[(4-azidophenyl)methylcarbonylamino] ethylaminocarbonyl]ethyl]phenyl]ethylamino-5'-N-ethylcarboxamidoad enosine (125I-azido-PAPA-APEC), and (c) partial purification of the hexahistidine-tagged receptor on Ni2+.nitrilotriacetic acid resin. A presumed A2a receptor (44 kDa) from rabbit striatal membranes also was detected with the antisera against amino acids Tyr155-Val172 of the RDC8 receptor. Not only could the mutant A2a receptor be photolabeled specifically with 125I-azido-PAPA-APEC but so too could unglycosylated A2a receptors (i.e., from tunicamycin-treated COS M6 cells), either full length or truncated. In all of these cases, photolabeling was attenuated by both agonist and antagonist competitors.

125I-2-[4-[2-[2-[(4-azidophenyl)methylcarbonylamino] ethylaminocarbonyl]ethyl]phenyl] ethylamino-5'-N-ethylcarboxamidoadenosine labels transmembrane span V of the A2a adenosine receptor.

We have shown previously that 125I-2-[4-[2-[2-[(4-azidophenyl)methylcarbonylamino] ethylaminocarbonyl]ethyl]phenyl] ethylamino-5'-N-ethylcarboxamidoadenosine (125I-azido-PAPA-APEC) specifically and selectively photolabels RDC8 A2a adenosine receptors that have been overexpressed in COS M6 cells. Glycosylated, 125I-azido-PAPA-APEC-labeled, wild-type (412 residues; 45,031 Da) and carboxyl-terminally truncated (315 residues; 35,427 Da) receptors migrate with apparent molecular masses of > 40 and 31.5 kDa, respectively, whereas unglycosylated or deglycosylated wild-type and truncated A2a receptors migrate with apparent molecular masses of 40 and 28.5 kDa, respectively. Because nonspecific photoincorporation is not a complication, the present peptide mapping studies of the full length and truncated canine A2a adenosine receptors were carried out on unpurified COS M6 membrane preparations. After partial proteolysis it became clear that glycosylation increased the apparent molecular mass of either the wild-type or mutant A2a receptor by approximately 3 kDa. Although the A2a receptor was readily cleaved by a variety of chemical reagents and proteases, trypsin and endoproteinase Glu-C generated the most reproducible and, in the case of trypsin, the most complete fragmentation patterns. Radiolabeled peptides were identified by their apparent molecular masses, (in)abilities to be recognized by an antipeptide antibody to amino acids Tyr155-Val172 of the presumed second extracellular loop of the receptor, and (in)sensitivities to endoglycosidase F and tunicamycin treatments. A prominent, 7-kDa, radiolabeled peptide that was generated by trypsin digestion implicated putative alpha-helix V in the binding of 125I-azido-PAPA-APEC.

Introduction of a putative dopaminergic prodrug moiety into a 6,7-substitution pattern characteristic of certain 2-aminotetralin dopaminergic agonists.

On the basis of the premise that the dopaminergic agonist profile of 2-(di-n-propylamino)-5-hydroxy-6-methyltetralin (1a) is due to in vivo oxidation of the 6-methyl moiety and that 1a may represent a novel prodrug strategy, the vicinal methyl-hydroxyl substitution pattern was incorporated into the 6- and 7-positions of 2-(di-n-propylamino)tetralin to give the 6-methyl-7-hydroxy and 6-hydroxy-7-methyl isomers 8 and 9, respectively. A multistep synthetic approach was devised which permitted preparation of target molecules 8 and 9. Pharmacological data revealed that both target compounds exhibit modest dopamine-like effects in the cardioaccelerator nerve assay in the cat, but neither appeared to be metabolically activated as was the case with 1a. The effects of 9 (but not of 8) were antagonized by pretreatment with haloperidol. Thus, the 5-hydroxy-6-methyl substitution pattern in the 2-aminotetralins remains unique as a dopaminergic agonist prodrug structure.