Anti-mu opioid antiserum against the third external loop of the cloned mu-opioid receptor acts as a mu receptor neutral antagonist.

The region from the third external loop to the C terminus of MOR-1 appeared to be critical to the selective binding of MOR-1 ligands as DAMGO and morphine to MOR-1. To study the pharmacological properties of the third extracellular loop an antibody was raised in rabbits against the sequence 304–316 which is unique to MOR-1 and includes the third external loop; the anti-MOR-1 antibody was affinity purified against the immunogen sequence and characterized by [3H]DAMGO andWestern blotting; [3H]DPDPE binding assay remained unchanged in the presence of the antibody. Anti-MOR-1 IgG was characterized as a neutral antagonist in Chinese hamster ovary (CHO) cells hyperexpressing constitutively active MOR-1s; in fact, anti-MOR-1 IgG completely reversed the inhibition induced by the MOR-1 agonist endomorphin1, endomorphin2, DAMGO and morphine on forskolin stimulated cyclic AMP (cAMP) accumulation and attenuated both the action of the selective MOR-1 agonistDAMGOto increase [35S]GTPgS binding and the action of the MOR-1 inverse agonist beta-chlornaltrexamine (CNA) to decrease [35S]GTPgS binding. Radioligand binding assay using membrane suspensions from CHO cells hyperexpressing MOR-1 revealed a significant decreased binding affinity and capacity of all the tested MOR-1 selective ligands after preincubation with anti-MOR-1 IgG. Therefore, the third extracellular loop of MOR-1 appeared to be a key element for the binding of MOR-1 ligands.