Opioid Receptors in Magnesium‐Digitonin‐Solubilized Rat Brain Membranes Are Tightly Coupled to a Pertussis Toxin‐Sensitive Guanine Nucleotide‐Binding Protein

Abstract

Opioid receptors solubilized in Mg2+-digitonin (2%, wt/vol) from Mg2+-pretreated rat brain membranes maintain, in addition to high-affinity opioid agonist binding, the modulation by guanine nucleotides. One of the modes of expression of the latter property is an attenuation of agonist binding by guanine nucleotides in the presence of Na+. To investigate the molecular basis of this modulation and to identify the G protein(s) involved, the soluble receptors were [32P]ADP-ribosylated by means of Bordetella pertussis toxin and subjected to molecular size exclusion chromatography. In addition, soluble extracts were chromatographed on lectin and hydrophobic affinity columns. The binding of 35S- and 3H-labelled analogues of GTP was also monitored in the species separated. The oligomeric G protein-coupled opioid receptors and the guanine nucleotide/pertussis toxin-sensitive species showed similar chromatographic properties in all three systems. This indicates that the biochemically functional G protein-opioid receptor complex formed in Mg2+-pretreated membranes in the absence of an agonist is stable in digitonin solution and to chromatographic separation. Further analysis showed that the guanine nucleotide modulation of opioid receptors is via the pertussis toxin substrates with Mr of 41,000 and 39,000, which are identified as Gi and Go alpha subunits, respectively.

Alkylation with β‐funaltrexamine suggests differences between μ‐opioid receptor systems in guinea‐pig brain and myenteric‐plexus

“…The popioid receptor is a G-protein coupled receptor (for reviews see Gilman, 1987;Milligan, 1988). Homogenization in Tris buffers generates a tightly coupled high agonist affinity opioid receptor (Wong et al, 1989). Addition of Na' ions and Gpp(NH)p restores the receptor to a form susceptible to alkylation by fl-FNA and therefore presumably similar to the form present in intact tissue.…”

“…The reasons for this discrepancy are unknown, but could for example relate to the composition of Krebs solution used, since ions are known to have marked effects on binding properties at opioid receptors (Paterson et al, 1986;Kosterlitz et al, 1987 Gilman, 1987;Milligan, 1988). Homogenization in Tris buffers generates a tightly coupled high agonist affinity opioid receptor (Wong et al, 1989 (Werling et al, 1988) and Na' ions and guanine nucleotides are known to shift the opioid receptor to a conformational state which is recognised by agonists with low affinity. However antagonist affinity is not reduced (Pert & Snyder, 1974;Simon et al, 1975;Blume, 1978 ibly (Portoghese & Takemori, 1983).…”

Regulation of Multiple Effectors by the Cloned δ‐Opioid Receptor: Stimulation of Phospholipase C and Type II Adenylyl Cyclase

“…The diverse signaling pathways utilized by the different opioid receptors share a common feature, namely, the involvement of G proteins that can be ADP-ribosylated by pertussis toxin (PTX) (Wong et al ., 1988(Wong et al ., , 1989Childers, 1991) . Recent studies revealed that the opioid receptors may have the ability to regulate many more effectors.…”

Go mediates the coupling of the mu opioid receptor to adenylyl cyclase in cloned neural cells and brain.

“…Purification of opioid receptor from rat brain yielded complexes containing either Gi or Go (16) and, in the NG108-15 neuroblastoma x glioma hybrid cells, the 8 receptor interacted with multiple subtypes of G proteins, including Gi2, Go (17,18), and Gi3 (18). Although these studies demonstrate the capability of opioid receptors to couple to various G proteins, they do not identify those interactions that transduce the signal to a given effector.…”