Abstract
To elucidate which portions of the opioid receptor molecules are involved in the ligand selectivity, we have expressed chimeric receptors between the rat delta- and mu-opioid receptors from cDNAs and analysed their ligand binding properties. We demonstrate that the major binding determinant for the delta-selective enkephalin-related peptide, [D-Pen2,D-Pen5]enkephalin, resides within the region comprising the transmembrane segments V-VII and the intervening loop regions. On the other hand, the region spanning from the intracellular loop I to the amino-terminal half of the transmembrane segment III is shown to be involved in determining high-affinity binding of the mu-selective enkephalin-related peptides, [D-Ala2, MePhe4,Gly-ol5]enkephalin and [D-Ala2,MePhe4,Met-ol5]enkephalin, whereas the major determinant for binding of the mu-selective alkaloids, morphine and codeine, is demonstrated to exist in the region spanning the transmembrane segments V-VII. These results indicate that distinct regions of the opioid receptor determine the selectivity for the delta- and the mu-selective enkephalin-related peptides and that the binding determinant for the mu-selective alkaloids is distinct from that for the mu-selective enkephalin-related peptides.
Highlights
In the BA series chimeras, the most remarkable change in the affinity to DAMO was found between BAI and BA2. These results suggest that the region spanning from intracellular loop (ICL) I to the amino-terminal half ofTM III determine the high-affinity binding of the JL-selective enkephalin-related peptides
The high-affinity binding of DPDPE, all-selective enkephalin-related peptide, was shown to be determined mainly by the region spanning TM V-VII of the Il-receptor and partly by the region spanning from the carboxyl-terminal half of TM III to extracellular loop (ECL) II and the carboxyl-terminal cytoplasmic region
The region spanning from ICL I to the amino-terminal half ofTM III of the jL-receptor was demonstrated to contain the major determinant for selectivity to DAGO and DAMO, jL-selective enkephalinrelated peptides
Summary
Construction of cDNAs Encoding Chimeric Receptors-Chimeric 01J.L cDNAs were constructed by exchange of restriction fragments from the cDNAs encoding the rat 0- and u-opioid receptors [7]. The restriction sites used and their positions in the deduced amino acid sequences of the o-receptorlJ.L-receptor, respectively, were as follows (Fig. 1):Acc!, at. The entire protein-coding sequences of the wild-type and chimeric cDNAs were inserted into the HindUI site of an expression vector pKCRH2 [17]. 10% fetal bovine serum were transfected by the calcium phosphate method [18] with expression vectors containing the wild-type or chimeric cDNAs. After 48-72-h incubation at 37°C, crude membrane was prepared from the transfected cells. 1,000 X g for 10 min and the precipitate suspended in the same buffer, homogenized, and centrifuged at 1,000 X g for 10 min. The apparent dissociation constants (Kd ) for opioid ligands were obtained by displacement of [3HJEKC binding by unlabeled ligands in the presence of 10 nM [3HJEKC and calculation by the equation Kd = IC501(1 +.
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