Site of pertussis toxin-induced ADP-ribosylation on Gi is critical for receptor modulation of GDP interaction with Gi

Abstract

In this study, the influence of the inhibitory μ-opioid receptor on the potencies of 5′-guanosine α-thio-triphoshate (GTPγS) and GDP at the inhibitory GTP-binding protein (Gi) were investigated in an adenylyl cyclase system. It was hoped that a receptor-mediated change in the potency of either GTPγS or GDP in affecting adenylyl cyclase activity may elucidate how a receptor alters cyclase activity via its G-protein. In an adenylyl cyclase system employing 5′-adenylyl imidodiphosphate as substrate, GTPγS, a nonhydrolyzable analog of GTP, inhibited forskolin-stimulated adenylyl cyclase activity in the absence of morphine; morphine failed to significantly affect the apparent potency of GTPγS. GDP blocked the GTPγS-induced inhibition of adenylyl cyclase; morphine profoundly diminished the ability of GDP to block the inhibitory effect of GTPγS. The IC50 values of GTPγS were 0.02 ± 0.01, 0.18 ± 0.04, and 2.2 ± 0.5 μM in the absence of other drugs, in the presence of a combination of 100 μM GDP and morphine, and in the presence of 100 μM GDP, respectively. GDP blocked the inhibitory effect of GTPγS (0.3 μM) in a concentration-dependent manner; the EC50 for GDP was 16 ± 2.6 μM in the absence of morphine and 170 ± 32 μM in the presence of morphine. Exposure of 7315c cells to pertussis toxin for 3 h resulted in a small decrease in the potency of GTPγS in inhibiting cyclase. However, the relative potency of GDP in blocking the GTPγS-mediated inhibition of cyclase was increased: the EC50 values of GDP were 11 ± 4 and 0.81 ± 0.2 μM in untreated and pertussis toxin-treated membranes, respectively. In untreated membranes, there was a brief lag in the GTPγS-induced inhibition of adenylyl cyclase; morphine diminished this lag. In membranes treated with pertussis toxin, there was an exaggerated lag in the onset of GTPγS inhibition of adenylyl cyclase activity; morphine could no longer affect this lag. Thus, uncoupling the μ-opioid receptor from Gi appeared to increase the affinity of Gi for GDP. These data suggest that the effect of an inhibitory receptor is to decrease the affinity of Gi for GDP by virtue of its interaction with the carboxy-terminal region of Giα. Since intracellular concentrations of GTP and GDP are both approximately 100 μM, occupancy of the μ-opioid receptor by an agonist would greatly increase the chances that GTP, rather than GDP, would interact with and activate Gi. © 1989 by The Endocrine Society.