Separation of acute desensitization and long-term tolerance of m-opioid receptors is determined by the degree of C-terminal phosphorylation

Phosphorylation of sites on the C terminus of the m-opioid receptor (MOR) results in the induction of acute desensitization that is thought to be a precursor for the development of long-term tolerance. Alanine mutations of all 11 phosphorylation sites on the C terminus of MORs almost completely abolished desensitization and one measure of tolerance in locus coeruleus neurons when these phosphorylation-deficient MORs were virally expressed in MOR knockout rats. In the present work, we identified specific residues that underlie acute desensitization, receptor internalization, and tolerance and examined four MOR variants with different alanine or glutamate mutations in the C terminus. Alanine mutations in the sequence between amino acids 375 and 379 (STANT-3A) and the sequence between amino acids 363 and 394 having four additional alanine substitutions (STANT + 7A) reduced desensitization and two measures of long-term tolerance. After chronic morphine treatment, alanine mutations in the sequence between 354 and 357 (TSST-4A) blocked one measure of long-term tolerance (increased acute desensitization and slowed recovery from desensitization) but did not change a second (decreased sensitivity to morphine). With the expression of receptors having glutamate substitutions in the TSST sequence (TSST-4E), an increase in acute desensitization was present after chronic morphine treatment, but the sensitivity to morphine was not changed. The results show that all 11 phosphorylation sites contribute, in varying degrees, to acute desensitization and long-term tolerance. That acute desensitization and tolerance are not necessarily linked illustrates the complexity of events that are triggered by chronic treatment with morphine. SIGNIFICANCE STATEMENT In this work, we showed that the degree of phosphorylation on the C terminus of the m-opioid receptor alters acute desensitization and internalization, and in measures of long-term tolerance to morphine. The primary conclusion is that the degree of phosphorylation on the 11 possible sites of the C terminus has different roles for expression of the multiple adaptive mechanisms that follow acute and long-term agonist activation. Although the idea that acute desensitization and tolerance are intimately linked is generally supported, these results indicate that disruption of one phosphorylation cassette of the C terminus TSST (354–357) distinguishes the two processes.