Studies on mu and delta opioid receptor selectivity utilizing chimeric and site-mutagenized receptors.

Abstract

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...

Novel “Restoration of Function” Mutagenesis Strategy to Identify Amino Acids of the δ-Opioid Receptor Involved in Ligand Binding

“…This protein binds nonselective opioid ligands but is devoid of affinity for ␦-selective ligands. Our results are in agreement with results from previous studies using /␦ or ␦/ chimeric receptors that have shown that ␦-selective ligands interact mainly with the region containing the sixth transmembrane domain and the third extracellular loop of the ␦-opioid receptor (35)(36)(37)(38)(39)(40)50). In this study, we have delimited this region to 10 amino acids that are located between arginine residue at position 291 and leucine residue at position 300.…”

“…Construction of the ␦/ 291-300 Chimera and Random Mutagenesis-Evidence from different groups has suggested that the third extracellular loop of ␦-opioid receptors is involved in the binding of selective ligands (35)(36)(37)(38)(39)(40)50). A chimeric ␦-opioid receptor in which 10 amino acids of the third extracellular loop (amino acids 291 to 300) were replaced by the corresponding amino acids from the receptor was constructed.…”

Moving from the Orphanin FQ Receptor to an Opioid Receptor Using Four Point Mutations

“…This is very similar to the behavior of the DynA peptides toward the receptor, suggesting that the C-terminal tail of the DynA fragments may be largely responsible for their limited affinity toward the Orphanin FQ receptor. Using the message-address concept (7,23) as applied to the opioid peptides, we can see that multiple messages exist in the C-terminal half of DynA- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). One of these messages, DynA-(8 -13), promotes the binding with the Orphanin FQ receptor; however, another message, found in the DynA-(14 -17) region, appears to discriminate against the Orphanin FQ receptor as evidenced by the observation that DynA-(1-13) has better affinity than DynA-(1-17) toward the Orphanin (but not the ) receptor.…”

“…To date, the body of evidence suggests that some specific residues near the interface of the extracellular loop and the ␣-helical transmembrane domains may play important roles in ligand binding through ionic or hydrogen bonding interactions (8 -10). The extracellular loops, which are highly divergent, clearly play a role in ligand selectivity, especially for the peptides, allowing them to differentiate between the , , and ␦ receptors (11)(12)(13)(14)(15). However, most of the mutagenesis studies aiming at defining a common opioid binding pocket rely on loss of binding as an indicator of a potential role of a given residue in ligand interaction.…”

“…The endogenous ligand for this opioid-like orphan receptor has recently been isolated by two independent groups (3,4). Interestingly, it is a 17-amino acid peptide with a significant degree of sequence homology to DynA- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). This novel peptide has been termed Nociceptin by Meunier et al (3) to denote its ability to increase pain responsiveness and Orphanin FQ by Civelli and coworkers (4).…”