Functional analysis of MOR-1 splice variants of the mouse mu opioid receptor gene Oprm.

Abstract

A series of mu opioid receptor gene Oprm splice variants have been reported that differ only at their C-terminus. These variants all contain exons 1, 2, and 3 of the gene, the exons responsible for coding all seven transmembrane domains. Whereas MOR-1 also has exon 4 that encodes for an additional 12 amino acids at the tip of the C-terminus, the other MOR-1 variants have unique amino acid sequences distinct from those in MOR-1 due to alternative splicing. All these variants are mu-selective in binding assays. The current study explored the ability of these variants to stimulate [35S]GTPgammaS binding to assess them functionally. Only mu opioids stimulated [35S]GTPgammaS binding. Among the mu opioids we noted marked differences in their maximal stimulation among the clones. This was most prominent with beta-endorphin, which stimulated [35S]GTPgammaS binding in the MOR-1E expressing cells to a greater degree than [D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAMGO; 130%) and was far less effective than DAMGO in MOR-1C cells (44%). The rank order of maximal stimulation of the drugs varied among the clones as well. Dynorphin A, beta-endorphin and morphine were most effective in stimulating [35S]GTPgammaS binding in MOR-1E, while M6G and fentanyl were most effective in MOR-1 expressing cells. The potency (EC50) of some of the drugs also varied extensively among the clones, with a poor correlation between the potency of the drugs to stimulate [35S]GTPgammaS binding and their binding affinity. Together, these findings reveal marked functional differences among the variants that only can be explained by their structural differences at the tip of their C-terminus.