DISTINCT DIFFERENCES BETWEEN MORPHINE- AND -ALA(2),N-MEPHE(4),GLY-OL(5)]-ENKEPHALIN-MU-OPIOID RECEPTOR COMPLEXES DEMONSTRATED BY CYCLIC-AMP-DEPENDENT PROTEIN-KINASE PHOSPHORYLATION

The present study demonstrates a conditional, agonist-dependent phosph orylation of the mu-opioid receptor (MOR-1) by cyclic AMP-dependent pr otein kinase (PKA) in membrane preparations of MOR-1-transfected neuro blastoma Neuro2A cells. Opioid agonist-dependent phosphorylation occur s in a time- and concentration-dependent manner (EC50 similar to 40 nM ) and can be abolished by the receptor antagonist naloxone. Stoichiome tric analysis indicates incorporation of a maximum of 6 mol of phospha te/mol of receptor in the presence of 1 mu M morphine and 6 nM PKA, Al though morphine and related alkaloids as well as some peptide agonists (PLO17 and beta-endorphin) stimulated phosphorylation of MOR-I by PKA , the potent mu-opioid-selective peptide [D-Ala(2),N-MePhe(4), Gly-ol( 5)]-enkephalin (DAMGO) or other enkephalin analogues such as [D-Ala(2) ] -Met(5)-enkephalinamide (DALA), [D-Ala(2),D-Leu(5)] -enkephalin (DAD LE), and Met(5)-enkephalin had no effect. The lack of the effect of DA MGO on MOR-1 phosphorylation state was evident also after chronic pret reatment. These results suggest the existence of different agonist-dep endent conformations of MOR-1. Furthermore, phosphorylation may be a u seful parameter with which to identify different agonist-receptor conf ormations.