Naloxone Activation of μ-Opioid Receptors Mutated at a Histidine Residue Lining the Opioid Binding Cavity

Abstract

The mu-opioid receptor is the principal site of action in the brain by which morphine, other opiate drugs of abuse, and endogenous opioid peptides effect analgesia and alter mood. A member of the seven-transmembrane domain (TM) G protein-coupled receptor (GPCR) superfamily, the mu-opioid receptor modulates ion channels and second messenger effectors in an opioid agonist-dependent fashion that is reversible by the classic opiate antagonist naloxone. Mutation of a histidine residue (His297) in TM 6 afforded agonist-like G protein-coupled signal transduction mediated by naloxone and other alkaloid antagonists and enhanced the intrinsic activity of documented alkaloid partial agonists, including buprenorphine. The intrinsic activities of all opioid peptide agonists and antagonists tested were not altered at the His297 mutant receptors. Consistent with a role for the TM 6 histidine in maintaining high affinity binding sites for opioid agonists and antagonists, opioid ligand-dependent protection of this residue from a histidine-specific alkylating agent indicated that the His297 side chain is positioned in or very near the binding cavity. The TM 6 His297 mutants identify a discrete region of the receptor critical for determining whether a specific drug pharmacophore triggers receptor activation. Because many GPCRs possess a similarly positioned TM histidine residue, our findings with the mu-opioid receptor may extend to these receptors and potentially serve as a model for rational design of therapeutic GPCR partial agonists and antagonists.

Molecular Basis for the Selectivity of the Mammalian Bombesin Peptide, Neuromedin B, for Its Receptor

“…This result is similar to the peptide agonist PL017 for the -opioid receptor where a [H297Q] receptor replacement altered affinity 5-fold (Spivak et al, 1997). To provide possible insight into the molecular basis of this change, we made several GRPR (position 202) point mutants.…”

Identification of new gonadotrophin-releasing hormone partial agonists

“…at the exoloop 2 or intraloop 2, which share 76 and 62% amino acid identity with the corresponding domains in the rGnRHR); in this setting, the particular structure of the rGnRHR antagonist might ultimately determine a preference for binding to the active receptor conformation of the hGnRHR and to both the inactive and active conformations of the rat counterpart, as would be expected for antagonist analogues. In fact, in a number of G protein-coupled receptors, including the GnRHR, mutations or species-dependent structural differences in either transmembrane domains (Strader et al 1989, Claude et al 1996, Noda et al 1996, Groblewski et al 1997, Han et al 1997, Spivak et al 1997, intraloops (Morin et al 1998, Wurch et al 1999 or exoloops (Sun et al 2001) may be limiting, to a variable extent, the level of maximal stimulation. It should be emphasized, however, that the effects of the compounds tested may be cell-specific and that depending on the relative levels of receptor, G proteins and other downstream effectors present in a particular tissue in vivo (i.e.…”

Molecular recognition of opioid receptor ligands

“…14 On the other hand, conservative mutations (ie, those that do not disrupt hydrogen bonding interactions, eg, histamine or glutamine to asparagine), had only minor effects on ligand binding. 15 From these results, it was suggested that His VI:17 hydrogen-bonds to the opioids ' phenol.…”