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Opioid receptors are members of the guanine nucleotide binding protein (G protein)-coupled receptor family. Three types of opioid receptors have been cloned and characterized and are referred to as the 6, ic, and ,u types. Analysis of receptor chimeras and site-directed mutant receptors has provided a great deal of information about functionally important amino acid side chains that constitute the ligand-binding domains and G-protein-coupling domains of G-protein-coupled receptors. We have constructed-6/,u opioid receptor chimeras that were expressed in human embryonic kidney 293 cells in order to define receptor domains that are responsible for receptor type selectivity. All chimeric receptors and wild-type 6 and ,u opioid receptors displayed high-affinity binding of etorphine (an agonist), naloxone (an antagonist), and bremazocine (a mixed agonist/antagonist).In contrast, chimeras that lacked the putative first extracellular loop of the ,u receptor did not bind the ,u-selective peptide MePhe4, The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.ceptor chimeras was used to focus site-directed mutagenesis to the third extracellular loop of the 8 opioid receptor. MATERIALS AND METHODSConstruction of Receptor Chimeras. Computer-aided alignment of the nucleotide sequences of the 8 and ,u opioid receptor cDNAs revealed six domains that have at least 15 identical contiguous nucleotides. These domains are located at the junction of the first intracellular loop and transmembrane domain 2 (TM2), at the junction of TM3 and the second intracellular loop, and in TM3, -5, -6, and -7. Junction sites used in the present studies are shown in Fig. 1. Pairs of complementary oligodeoxyribonucleotides were synthesized (Operon Technologies, Alameda, CA) corresponding to these homologous domains and had the following sequences (written in the 5' to 3' direction): TM2+, GCCACCAACATCTACAT; TM2-, ATGTAGATGTTGGTGGC; TM3+, TACTACAA-CATGTTCAC; TM3-, GTGAACATGTTGTAGTA; TM5 +, TGCCGATCCTCATCATCAC; TM5-, GTGAT-GATGAGGATCGGCA; TM6+, ATGGTGCTGGTGGTC/ GGTG; TM6-, CACG/CACCACCAGCACCAT; TM7+, GTTCTTTACGCCTTCCTGG; TM7-, CCAGGCAGGCG-TAAAGAAC. Expression plasmids encoding receptor chimeras were constructed by using a two-step recombinant PCR protocol (Fig. 2). Template DNAs were the 8 receptor cDNA (4) that we had subcloned into the vector pCR3 (Invitrogen) and the ,u receptor cDNA (7) that we subcloned into the pRc/CMV vector (Invitrogen). The pCR3 and pRc/CMV vectors contain T7 and Sp6 RNA polymerase promoters upstream and downstream of the receptor cDNA inserts, respectively, and oligodeoxyribonucleotides corresponding to these promoters were used in combination with the TM+/-receptor oligodeoxyribonucleotides to generate the primary PCR fragments. An aliquot from each primary PCR was analyzed by agarose gel electrophoresis to estimate yield and specificity, and then aliquots were an...

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The sensitivity of and ␦ receptor binding to dithiothreitol and N-ethylmaleimide was examined to probe receptor structure and function. Binding to both receptor types was inhibited by dithiothreitol (IC 50 values ‫؍‬ 250 mM), suggesting the presence of inaccessible but critical disulfide linkages. receptor binding was inhibited with more rapid kinetics and at lower N-ethylmaleimide concentrations than ␦ receptor binding. Ligand protection against N-ethylmaleimide inactivation suggested that alkylation was occurring within, or in the vicinity of, the receptor binding pocket. Sodium ions dramatically affected the IC 50 of N-ethylmaleimide toward both receptor types in a ligand-dependent manner. Analysis of receptor chimeras suggested that the site of N-ethylmaleimide alkylation on the receptor was between transmembrane domains 3 and 5. Substitution of cysteines between transmembrane domains 3 and 5 and elsewhere had no effect on receptor binding or sensitivity toward N-ethylmaleimide. Serine substitution of His 223 in the putative second extracellular loop linking transmembrane domains 4 and 5 protected against Nethylmaleimide inactivation. The H223S substitution decreased the affinity of bremazocine 25-fold, highlighting the importance of this residue for the formation of the high affinity bremazocine binding site in the opioid receptor.Three major types of opioid receptor, ␦, , and , have been cloned and characterized extensively (reviewed in Refs. 1 and 2). There is approximately 60% amino acid sequence identity between the opioid receptor types. The ␦, , and opioid receptors have unique ligand specificities, anatomical distributions, and physiological functions (3). Morphine, related opioid drugs, and the endogenous opioid peptides activate signal transduction pathways by binding to opioid receptors (4), which are members of the G protein-coupled receptor family (5). G protein-coupled receptors are seven-transmembrane domain (TM) 1 proteins that mediate signal transduction across the plasma membrane. The ligands approach and engage the receptor from the extracellular side, and receptor activation results in the coupling to heterotrimeric G proteins on the intracellular face of the membrane. Opioid receptor types interact with multiple G proteins (6 -8) to regulate adenylyl cyclase, Ca 2ϩ channels, and K ϩ channels. It has been known from early studies on the characterization of opioid receptors that specific binding is inhibited by sulfhydryl reagents, such as iodoacetamide, N-ethylmaleimide (NEM), and p-hydroxymercuribenzoate (9 -11). Preincubation with opioid ligands protected against receptor inactivation, suggesting that the sensitive sulfhydryl group was located within or near the binding site. Evidence has been obtained that analogs of Leu-enkephalin and morphine, containing activated sulfhydryl groups, form mixed disulfide linkages with opioid receptors (12, 13). The covalently bound agonists caused receptor activation that persisted following extensive washing, yet was naloxone-reversible. The result...

The polymorphism in a DNA 16-mer (designated BZ-II) has been investigated by means of circular dichroism (CD) spectroscopy and chemical footprinting. CD spectra indicate that, in low salt, the oligomer is fully right-handed whereas, in high salt, it possesses a B-Z conformational junction: half of the duplex is right-handed while the other half is left-handed. Treatment of BZ-II with diethyl pyrocarbonate (DEPC) confirms the existence of a left-handed segment of the duplex in high salt: enhanced DEPC scission occurs at the G residues in the alternating CG sequence. The scission patterns of the upper and lower strands in BZ-II by the reactive chemical probe MPE.Fe(II), and the antitumor antibiotics dynemicin and Fe-(II).bleomycin, are different under low salt conditions. The 3'-terminal region of both upper and lower strands and the middle region of the upper strand of BZ-II are preferential cleavage sites in low salt. This result suggests that the methylated cytosines or the alternating CG domain in the molecule perturbs the DNA structure. Under high salt conditions, the reactivity of the Z-DNA segment of BZ-II for MPE.Fe(II) and Fe(II).bleomycin is dramatically enhanced, while it is reduced in the case of dynemicin. Excess propidium (PI) eliminates preferential cleavage by each of these chemical probes in high salt conditions. This is due in part to conversion of the BZ-DNA molecule into B-DNA, as is seen by a DEPC modification experiment.(ABSTRACT TRUNCATED AT 250 WORDS)