A common single nucleotide polymorphism A118G of the μ opioid receptor alters its N-glycosylation and protein stability

Abstract

Synopsis The A118G single nucleotide polymorphism (SNP) of the human mu opioid receptor (hMOPR) gene OPRM1 results in an amino acid substitution (N40D). Subjects homozygous for G118 allele were reported to require higher morphine doses to achieve adequate analgesia and G118 allele was more prevalent among drug abusers. However, changes in the MOPR protein associated with this SNP are unknown. Using a knock-in mouse model (G/G mice) that possesses the equivalent nucleotide/amino acid substitution (N38D; A112G) of the A118G in the hMOPR gene , we investigated N-linked glycosylation status of thalamic and striatal MOPR in G/G mice vs A/A (wildtype) mice. The relative molecular mass (Mr) of MOPR determined with immunoblotting was lower in G/G mice than in A/A mice. Following treatment with PNGase F, which removes all N-linked glycans, both MOPR variants had identical Mr, indicating that this discrepancy was due to a lower level of N-glycosylation of the MOPR in G/G mice. In CHO cells stably expressing hMOPRs, G118/D40-hMOPR had lower Mr than A118/N40-hMOPR, which was similarly due to differential N-glycosylation. Pulse-chase studies revealed that the half-life of the mature form of G118/D40-hMOPR (∼12h) was shorter than that of A118/N40-hMOPR (∼28h). Thus, A118G SNP reduces MOPR N-glycosylation and protein stability.

How Much Oxycodone Is Needed for Adequate Analgesia After Breast Cancer Surgery: Effect of the OPRM1 118A>G Polymorphism

“…34 This amino acid change alters the binding potential and half-life of the receptor and thereby modulates the downstream functions of the receptor. 2,6,13 Also contradictory findings exist and it seems possible that the molecular consequences of this polymorphism are cell or tissue type specific. 34 As 118A>G has the highest allelic frequency of all amino acid altering OPRM1 SNPs and the mu-opioid receptor conveys most of the opioid effects this polymorphism could significantly influence the response to opioid therapy.…”

“…This polymorphism leads to asparagine being replaced by aspartate, which changes the N-glycosylation of the protein. 2,13 The polymorphism is relatively common, having a frequency around 10-15% in the European populations, but there is also large interethnic variability in the allele frequency. 34 This amino acid change alters the binding potential and half-life of the receptor and thereby modulates the downstream functions of the receptor.…”

Labeling and Expression of Opioid Receptors Using N-terminal Fusion of Fluorescent Proteins

“…Cell surface expression was achieved but FP-MOR was limited to insect cells and receptors were observed to be trapped within intracellular compartments [7]. In mammalian cells it appeared that insertion of a signal sequence interfered with the natural regulation of expression via glycosylation [8,9] or alternate strategies such as binding to p24A through the acidic residues at the second extracellular loop [10].…”

Opioid pharmacogenetics of alcohol addiction

“…The A (asparagine) allele is thought to be N-glycosylated (Huang et al 2012), whereas this is not possible for the G (aspartate) allele, as there is no free amino group. Subsequent study (e.g., Gelernter et al 1999;Szeto et al 2001;Crowley et al 2003;Tan et al 2003) revealed large ethnic differences in allele frequencies (see Table 1).…”