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

Brain Res Mol Brain Res. 2003 Nov 6;119(1):100-10. doi: 10.1016/j.molbrainres.2003.08.019.

Abstract

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 and Western 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 agonist DAMGO to increase [35S]GTPgammaS binding and the action of the MOR-1 inverse agonist beta-chlornaltrexamine (CNA) to decrease [35S]GTPgammaS 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.

MeSH terms

  • Animals
  • Antibodies / pharmacology*
  • Binding Sites / drug effects
  • Binding Sites / immunology
  • Binding, Competitive / drug effects
  • Binding, Competitive / physiology
  • CHO Cells
  • Cell Membrane / drug effects
  • Cell Membrane / immunology
  • Cricetinae
  • Cyclic AMP / metabolism
  • Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / pharmacology
  • Guanosine 5'-O-(3-Thiotriphosphate) / pharmacology
  • Ligands
  • Male
  • Mice
  • Mice, Knockout
  • Protein Binding / immunology
  • Protein Structure, Tertiary / drug effects
  • Protein Structure, Tertiary / physiology
  • Rabbits
  • Receptors, G-Protein-Coupled / immunology*
  • Receptors, G-Protein-Coupled / metabolism*
  • Receptors, Opioid, mu / genetics
  • Receptors, Opioid, mu / immunology*
  • Receptors, Opioid, mu / metabolism*

Substances

  • Antibodies
  • Ligands
  • Receptors, G-Protein-Coupled
  • Receptors, Opioid, mu
  • Enkephalin, Ala(2)-MePhe(4)-Gly(5)-
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • Cyclic AMP