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.

Arg2074Cys missense mutation in the C2 domain of factor V causing moderately severe factor V deficiency: molecular characterization by expression of the recombinant protein.

Factor V (FV) deficiency is a rare bleeding disorder whose genetic basis has been described in a relatively small number of cases. Among a total of 12 genetic defects reported in severely or moderately severe deficient patients, 3 were missense mutations and in no case was the mechanism underlying the deficiency explored at the molecular level. In this study, a homozygous missense mutation at cDNA position 6394 in exon 23 of the FV gene was identified in a 22-year-old Italian patient. This mutation causes the replacement of arginine 2074 with a cysteine residue (Arg2074Cys) in the C2 domain of the protein. The effect of the Arg2074Cys mutation on FV secretion, stability, and activity was investigated. Site-directed mutagenesis of FV cDNA was used to introduce the identified mutation, and wild-type as well as mutant FV proteins were expressed by transient transfection in COS-1 cells. An enzyme immunoassay detected low FV antigen levels both in the conditioned media of cells expressing the mutant protein and in cell lysates. Metabolic labeling and pulse-chase experiments confirmed that the mutation caused an impaired secretion of FV associated with rapid intracellular degradation. In addition, evaluation of wild-type and mutant coagulant activity demonstrated that the FV molecules carrying the Arg2074Cys mutation have reduced activity. These findings, beside confirming the structural and functional importance of the arginine 2074 residue, demonstrate that its substitution with a cysteine impairs both FV secretion and activity.