G sα-dependent and -independent desensitisation of prostanoid IP receptor-activated adenylyl cyclase in NG108-15 cells

Abstract

NG108-15 mouse neuroblastoma X rat glioma cells were treated with the prostanoid IP receptor agonist iloprost (1 μM) and the time course of changes in the levels of prostanoid IP receptors, adenylyl cyclase activity, and the α-subunit of the stimulatory guanine nucleotide binding regulatory protein, G s, were measured. Incubation of cells with iloprost produced a biphasic time course of desensitisation of prostanoid IP receptor-activated adenylyl cyclase. Parallel analysis of iloprost-induced loss of membrane G sα, NaF-stimulated adenylyl cyclase and [ 3H]iloprost binding suggested only monophasic curves, with t 0.5 values similar to the initial phase of desensitisation of iloprost-stimulated adenylyl cyclase activity. This suggests that the loss of receptor and G sα occur at the same time and account for the initial period of desensitisation due to iloprost pretreatment. Pretreatment of NG108-15 cells with cholera toxin produced a near complete loss of membrane-associated G sα, but the loss of [ 3H]iloprost binding due to iloprost treatment was not affected by pretreatment with cholera toxin, suggesting that prostanoid IP receptors can be down-regulated in the absence of any coupling to G s. The second phase of desensitisation of iloprost-stimulated adenylyl cyclase activity, during which there was no further change in NaF-stimulated adenylyl cyclase or in the membrane levels of G sα, was not due to protein kinase A activation, since elevating intracellular cyclic AMP levels with forskolin did not subsequently decrease iloprost-stimulated adenylyl cyclase activity or [ 3H]iloprost binding. These results demonstrate the iloprost pretreatment of NG108-15 cells induces two distinct phases of desensitisation; an initial desensitisation due to concurrent loss of prostanoid IP receptors and G sα, and then a further desensitisation by an as yet uncharacterised mechanism during which there is no further loss of G sα.