Chronic morphine induces the concomitant phosphorylation and altered association of multiple signaling proteins: A novel mechanism for modulating cell signaling

Traditional mechanisms thought to underlie opioid tolerance include recepto r phosphorylation/down-regulation, C-protein uncoupling, and adenylyl cycla se superactivation. A parallel line of investigation also indicates that op ioid tolerance development results from a switch from predominantly opioid receptor G(i alpha) inhibitory to G(beta gamma), stimulatory signaling. As described previously, this results, in part, from the increased relative ab undance of G(beta gamma)-stimulated adenylyl cyclase isoforms as well as fr om a profound increase in their phosphorylation [Chakrabarti, S., Rivera, M ., Yan, S.-Z., Tang, W.-J. & Gintzler, A. R. (1998) Mel. Pharmacol. 54, 655 -662; Chakrabarti, S., Wang, L., Tang, W.-J. & Gintzler, A, R. (1998) Mel. Pharmacol. 54 949-953], The present study demonstrates that chronic morphin e administration results in the concomitant phosphorylation of three key si gnaling proteins, G protein receptor kinase (GRK) 2/3, beta -arrestin, and G(beta), in the guinea pig longitudinal muscle myenteric plexus tissue. Aug mented phosphorylation of all three proteins is evident in immunoprecipitat e obtained by using either anti-GRK2/3 or G(beta) antibodies, but the phosp horylation increment is greater in immunoprecipitate obtained with G(beta) antibodies. Analyses of coimmunoprecipitated proteins indicate that phospho rylation of GRK2/3, beta -arrestin, and G(beta) has varying consequences on their ability to associate. As a result, increased availability of and sig naling via G(beta gamma) could occur without compromising the membrane cont ent land presumably activity) of GRK2/3. Induction of the concomitant phosp horylation of multiple proteins in a multimolecular complex with attendant modulation of their association represents a novel mechanism for increasing G(beta gamma) signaling and opioid tolerance formation.