Opioid Receptors of Neuroblastoma Cells Are in Two Domains of the Plasma Membrane that Differ in Content of G Proteins

Publisher Website
Google Scholar
Abstract
Opioid receptors of NG 108–15 cell membranes are distributed in two membrane fractions sedimenting at 20,000 g (P2) and 200,000 g (P3). The number of receptors is identical in P2 and P3, but in P2 all sites are present in one high-affinity state (2 nM), whereas in P3 60% of these receptors display lower affinity (150 nM). Upon addition of GTP or pretreatment with pertussis toxin, 80% of the sites exist in low affinity in both P2 and P3. Therefore, the effect of GTP and pertussis toxin on agonist binding appears to be smaller in P2 than in P3. In contrast, sodium inhibits agonist binding in P2 and P3 to the same extent and with identical potency. Opioid-mediated stimulation of GTPase is much greater in P2 than in P3, whereas inhibition of adenylate cyclase does not differ in the two fractions. Using site-specific antibodies and pertussis toxin-catalyzed ADP-ribosylation, we found that the amount of G proteins in P3 is only 30–50% of that in P2. Treatment of intact cells with the hydrophilic protein-modifying agent sulfosuccinimido-biotin results in biotinylation of proteins from both fractions and in a similar reduction of opioid binding in P2 and P3. Likewise, exposure of intact cells to the alkylating opioid antagonist, chlornaltrexamine, produces identical degrees of receptor inactivation in P2 and P3. The rate of in vivo pertussis toxin-mediated modification of G proteins is not different in the two fractions. We conclude from these data that (1) a larger proportion of receptors is uncoupled from G proteins in P3 than in P2; (2) the effects of GTP and Na+ on opioid receptor binding can be attributed to different membrane components; and (3) receptors in P3 appear to be present in the plasma membranes and, thus, do not represent internalized receptors.