Multiple agonist-affinity states of opioid receptors: Regulation of binding by guanyl nucleotides in guinea pig cortical, NG108-15, and 7315c cell membranes

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Journal Article

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Molecular Pharmacology






Multiple affinity states of opioid receptors of the μ and δ types have been identified in membranes prepared from cells which bear only one type of opioid receptor (μ receptors in 7315c cells, δ receptors in NG 108-15 cells), and in guinea pig cortical membranes where both types of receptors were present in the membrane preparations. States of μ and δ receptors which have agonist affinities too low to be identified by radiolabeled agonist have been measured indirectly by agonist competition for sites labeled by radioactive antagonist. Using analogues of guanyl nucleotides, we have examined the competition of the μ and δ agonists DAGO and DSLET against [3H]DIP or [3H]NAL binding to opioid receptors and identified several agonist affinity states. In the absence of added nucleotide, competition of DSLET for [3H]DIP binding to δ opioid receptors revealed the presence of two binding sites with differing apparent agonist affinities. Addition of GDPβS produced a steep monophasic curve which was best fit by a one-site model. In contrast, in the presence of added GTP or GTPγS, two affinity states were again apparent for DSLET competition at the δ receptor. The competition curve with GTP was shifted to the right relative to that produced in the absence of added guanyl nucleotide, indicating the presence of a lower apparent affinity state than any observed under other treatment conditions. DAGO competed against [3H]DIP or [3H]NAL binding to μ receptors over a wide concentration range in the absence of added guanyl nucleotide, consistent with the occupation by this ligand of more than one agonist affinity state of the μ receptor. However, when GDPβS was added to the incubation mixture, only a single binding site was identified. Two μ receptor affinity states were again observed in the presence of added GTP or GTPγS. One of these had significantly lower apparent affinity than those states detected in the absence of added nucleotide or with GDPβS. Pertussis toxin treatment resulted in a monophasic agonist competition curve which was best fitted by a single-site model in both 7315c and NG108-15 cell membranes. Addition of 100 μM GTP did not affect the agonist K(app) or B(max) after pertussis toxin treatment, suggesting that sites labeled under these conditions were not functionally associated with a G protein. In general, the effects of guanyl nucleotides were qualitatively similar at μ and δ receptors. The multiple apparent affinity states of each type of receptor probably reflect the preferential occurrence of different forms of agonist-receptor-G protein-guanyl nucleotide complex depending on the agonist or antagonist properties of the ligand and the guanyl nucleotides present.

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