The μ-opioid receptor variant N190K is unresponsive to peptide agonists yet can be rescued by small-molecule drugs.
The μ-opioid receptor (MOR) plays an important role in modulating analgesia, feeding behavior, and a range of autonomic functions. In the current study, we investigated the degree to which 13 naturally occurring missense mutations affect the pharmacological properties of the human MOR. After expression of each receptor in human embryonic kidney 293 cells, signaling (Gα(i/o)-mediated) induced by peptide agonists was assessed using luciferase reporter gene assays. Multiple mutants (S66F, S147C, R260H, R265C, R265H, and S268P) show a significant reduction in agonist potency. At the N190K variant, agonist-mediated signaling was essentially absent. Enzyme-linked immunosorbent assay, microscopic analysis, and radioligand binding assays revealed that this mutant shows markedly reduced cell-surface expression, whereas all other receptor variants were expressed at normal levels. Surface expression of the N190K variant could be increased by incubation with the alkaloid agonist buprenorphine or with either naltrexone or naloxone, structurally related MOR antagonists. We were surprised to find that both putative antagonists, despite being inactive at the wild-type MOR, triggered a concentration-dependent increase in N190K receptor-mediated signaling. In contrast, peptidic ligands failed to promote expression or rescue function of the N190K mutant. Subsequent analysis of the N190K variant in an ethnically diverse cohort identified this isoform in a subgroup of African Americans. Taken together, our studies reveal that the N190K mutation leads to severe functional alterations and, in parallel, changes the response to established MOR ligands. The extent to which this mutation results in physiological abnormalities or affects drug sensitivity in selected populations (e.g., those with chronic pain or addiction) remains to be investigated.
Fortin
Jean-Philippe
JP
Molecular Pharmacology Research Center, Molecular Cardiology Research Institute, Tufts Medical Center, Tufts University School of Medicine, Boston, Massachusetts, USA.
Ci
Lei
L
Schroeder
Jonathan
J
Goldstein
Carmit
C
Montefusco
Maria Claudia
MC
Peter
Inga
I
Reis
Steven E
SE
Huggins
Gordon S
GS
Beinborn
Martin
M
Kopin
Alan S
AS
D001741:Black or African American; D019943:Amino Acid Substitution; D002460:Cell Line; D008076:Cholesterol, HDL / Q000097:blood; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D005260:Female; D017930:Genes, Reporter; D005838:Genotype; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009270:Naloxone / Q000494:pharmacology; D009271:Naltrexone / Q000494:pharmacology; D009292:Narcotic Antagonists / Q000494:pharmacology; D018847:Opioid Peptides / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology*; D020641:Polymorphism, Single Nucleotide; D020033:Protein Isoforms / Q000819:agonists / Q000235:genetics; D021381:Protein Transport; D011869:Radioligand Assay; D017450:Receptors, Opioid, mu / Q000819:agonists* / Q000096:biosynthesis / Q000235:genetics; D015398:Signal Transduction; D044465:White People
D008076:Cholesterol, HDL; D009292:Narcotic Antagonists; D018847:Opioid Peptides; D010455:Peptides; D020033:Protein Isoforms; D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D009270:Naloxone; D009271:Naltrexone; D008156:Luciferases